Anti pd-l1 antibodies

ABSTRACT

The present disclosure relates to antibodies and antigen-binding fragments thereof that bind to PD-L1, and to methods of using such antibodies and antigen-binding fragments. For example, the present invention provides humanized anti-PD-L1 antibodies and methods of use thereof.

RELATED APPLICATIONS

This application is a Divisional application which claims the benefit ofU.S. application Ser. No. 15/328,232 filed Jan. 23, 2017, now U.S. Pat.No. 10,435,470, which is a U.S. National Stage of InternationalApplication No. PCT/US2015/043723, filed Aug. 5, 2015, which claimspriority to International Application No. PCT/CN2014/083715, filed Aug.5, 2014, each of which is incorporated herein by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen-bindingfragments thereof that bind to PD-L1, and to methods of using suchantibodies and antigen-binding fragments.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The content of the text file submitted electronically herewith isincorporated herein by reference in its entirety: A computer readableformat copy of the Sequence Listing (filename:CRBI_007_00WO_SeqList_ST25.txt); date recorded: Jul. 28, 2014; file size153 KB).

BACKGROUND

Programmed death receptor Ligand 1 (PD-L1) is a ligand of programmeddeath receptor 1 (PD-1). PD-1 is primarily expressed on lymphocytes andhas two ligands, PD-L1 and PD-L2. PD-L2 is not as common as PD-L1. PD-L1is also known as cluster of differentiation 274 (CD274) or B7 homolog 1(B7-H1) and is a 40 kDa type 1 transmembrane protein which is encoded bythe CD274 gene. Both PD-L1 and PD-1 belong to immunoglobulin superfamilyand consist of two extracellular Ig domains, an N-terminal V domain, anda C-terminal constant domain. The binding interface of PD-L1 toprogrammed death 1 (PD-1) and B7-1 (CD80) is on the IgV-like domain (Linet al. (2008) PNAS 105:3011-3016). While PD-L1 contains a conservedshort intracellular tail (about 30 amino acids), PD-1 contains twocytoplasmic tyrosine-based signaling motifs, an immunoreceptortyrosine-based inhibition motif (ITIM) and an immunoreceptortyrosine-based switch motif (ITSM). Following T cell stimulation, PD-1recruits the tyrosine phosphatase SHP-2 to the ITSM motif within itscytoplasmic tail, leading to the dephosphorylation of effector moleculessuch as CD3 Zeta, PKC theta and ZAP70 that are involved in the CD3 Tcell signaling cascade (Freeman et al. (2000) J Exp Med 192:1027-34;Latchman, et. al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur JImmunol 32:634-43). PD-L1 is not only widely distributed on leukocytesand nonhematopoietic cells in lymphoid and nonlymphoid tissues, but alsoin various cancer cells. Clinical data suggest that high tumorexpression of PD-L1 is associated with increased tumor aggressivenessand poorer prognosis. The formation of PD-1/PD-L1 complex transmits aninhibitory signal and negatively regulates T cell immune responses; itinhibits TCR-mediated T cell activation, cytokine production and T cellproliferation (Fife et al. (2011) Nature Immunology 10:1185-1193);induces exhaustion or anergy among cognate antigen-specific T cells(Hofmeyer et al. (2011) Journal of Biomedicine and Biotechnology2011:1-9); promotes the differentiation of Th1 cells into Foxp3+regulatory T cells (Armanath et al. (2011) Science TransMed 3:1-13;Francisco et al. (2009) J. Exp. Med. 206:3015-3029); and inducesapoptosis of effector T cells. Disruption of the PD-L1 gene leads toup-regulated T cell responses and the generation of self-reactive Tcells (Latchman et al. (2004) PNAS 101:10691-10696). Antibody blockadeof either PD-1 or PD-L1 leads to increased antitumor immunity (Iwai etal. (2002) PNAS 99:12293-12297).

Thus, there is an important role for the PD-1/PD-L1 pathway incontrolling immune responses. Dysfunction of PD-1/PD-L1 signalingappears to be correlated with initiation and development of diseasessuch as cancer and viral infection. Analysis of knockout animals has ledto the understanding that PD-1/PD-L1 functions mainly in inducing andregulating peripheral tolerance. Thus, therapeutic blockade of thePD-1/PD-L1 pathway would be helpful in overcoming immune tolerance andin the treatment of cancer or infection as well as in boosting immunityduring vaccination (either prophylactic or therapeutic). There is a needin the art for improved methods for blocking the PD-1/PD-L1 pathway.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides antibodies andantigen-binding fragments thereof that bind to programmed death-1 ligand1(PD-L1). In some embodiments, the antibodies and antigen-bindingfragments thereof bind to human PD-L1. In some embodiments, theantibodies and antigen-binding fragments thereof bind to PD-L1 and blockbinding of PD-1 and/or CD80 to PD-L1. In further embodiments, theanti-PD-L1 antibodies and fragments thereof bind to PD-L1 and disruptthe PD-L1/PD-1 or PD-L1/CD80 pathway. In one embodiment, the antibody orfragment thereof is a murine antibody, a chimeric antibody, a humanantibody or a humanized antibody. In one embodiment, the anti-PD-L1antibody or fragment thereof is a monoclonal antibody, scFv, Fabfragment, Fab′ fragment, F(ab)′ fragment, bispecific antibody,immunoconjugate, or a combination thereof

In one embodiment, the present invention provides an isolated antibodyor fragment thereof comprising one or more CDRs selected from the groupconsisting of SEQ ID NOs: 81-140.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR1 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:81, 87, 93, 99, 105, 111, 117, 123, 129, and 135.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR2 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:82, 88, 94, 100, 106, 112, 118, 124, 130, and 136.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR3 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:83, 89, 95, 101, 107, 113, 119, 125, 131, and 137.

In one embodiment, the antibody or fragment thereof comprises a lightchain CDR1 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:84, 90, 96, 102, 108, 114, 120, 126, 132, and 138.

In one embodiment, the antibody or fragment thereof comprises a lightchain CDR2 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:85, 91, 97, 103, 109, 115, 121, 127, 133, and 139.

In one embodiment, the antibody or fragment thereof comprises a lightchain CDR3 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:86, 92, 98, 104, 110, 116, 122, 128, 134, and 140.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR1 consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 81, 87, 93, 99, 105, 111, 117, 123, 129, and135; a heavy chain CDR2 consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 82, 88, 94, 100, 106, 112, 118,124, 130, and 136; a heavy chain CDR3 consisting of an amino acidsequences selected from the group consisting of SEQ ID NOs: 83, 89, 95,101, 107, 113, 119, 125, 131, and 137; a light chain CDR1 consisting ofan amino acid sequence selected from the group consisting of SEQ ID NOs:84, 90, 96, 102, 108, 114, 120, 126, 132, and 138; a light chain CDR2consisting of an amino acid sequence selected from the group consistingof SEQ ID NOs: 85, 91, 97, 103, 109, 115, 121, 127, 133, and 139 and alight chain CDR3 consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 86, 92, 98, 104, 110, 116, 122, 128,134, and 140.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 81, 82, and83, respectively; and a light chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:84, 85, and 86, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a heavy chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 81, 82, and 83, respectively, and a light chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 84, 85, and 86,respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 87, 88, and89, respectively; and a light chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:90, 91, and 92, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a heavy chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 87, 88, and 89, respectively, and a light chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 90, 91, and 92,respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 93, 94, and95, respectively; and a light chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:96, 97, and 98, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a heavy chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 93, 94, and 95, respectively, and a light chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 96, 97, and 98,respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 99, 100, and101, respectively; and a light chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:102, 103, and 104, respectively. In a further embodiment, the antibodyor antibody fragment thereof comprises a heavy chain CDR1, CDR2, andCDR3 according to SEQ ID NOs: 99, 100, and 101, respectively, and alight chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 102, 103, and104, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 105, 106,and 107, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 108, 109, and 110, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a heavy chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 105, 106, and 107, respectively,and a light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 108,109, and 110, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 111, 112,and 113, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 114, 115, and 116, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a heavy chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 111, 112, and 113, respectively,and a light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 114,115, and 116, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 117, 118,and 119, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 120, 121, and 122, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a heavy chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 117, 118, and 119, respectively,and a light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 120,121, and 122, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 123, 124,and 125, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 126, 127, and 128, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a heavy chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 123, 124, and 125, respectively,and a light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 126,127, and 128, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 129, 130,and 131, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 132, 133, and 134, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a heavy chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 129, 130, and 131, respectively,and a light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 132,133, and 134, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 135, 136,and 137, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 138, 139, and 140, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a heavy chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 135, 136, and 137, respectively,and a light chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 138,139, and 140, respectively.

In one embodiment, the antibody or fragment thereof binds PD-L1 andcomprises a heavy chain variable region comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46; and alight chain variable region comprising an amino acid sequence having atleast 80% homology, at least 85% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48. In a furtherembodiment, the isolated antibody or fragment thereof binds PD-L1 andcomprises a heavy chain variable region comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34,38, 42, and 46; and a light chain variable region comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36,40, 44, and 48.

In one embodiment, the invention provides anti-PD-L1 antibodies thatcomprise a variable heavy chain of an antibody selected from the groupconsisting of 13C5, 5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, and 15F1and a variable light chain of an antibody selected from the groupconsisting of 13C5, 5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, and 15F1.Thus, in one embodiment, the invention provides an antibody or fragmentthereof comprising a heavy chain variable region comprising SEQ ID NO: 2and a light chain variable region comprising SEQ ID NO: 4; a heavy chainvariable region comprising SEQ ID NO: 6 and a light chain variableregion comprising SEQ ID NO: 8; a heavy chain variable region comprisingSEQ ID NO: 10 and a light chain variable region comprising SEQ ID NO:12; a heavy chain variable region comprising SEQ ID NO: 14 and a lightchain variable region comprising SEQ ID NO: 16; a heavy chain variableregion comprising SEQ ID NO: 18 and a light chain variable regioncomprising SEQ ID NO: 20; a heavy chain variable region comprising SEQID NO: 22 and a light chain variable region comprising SEQ ID NO: 24; aheavy chain variable region comprising SEQ ID NO: 26 and a light chainvariable region comprising SEQ ID NO: 28; a heavy chain variable regioncomprising SEQ ID NO: 30 and a light chain variable region comprisingSEQ ID NO: 32; a heavy chain variable region comprising SEQ ID NO: 34and a light chain variable region comprising SEQ ID NO: 36; or a heavychain variable region comprising SEQ ID NO: 38 and a light chainvariable region comprising SEQ ID NO: 40.

In one embodiment, the present invention provides a chimeric anti-PD-L1antibody, wherein the antibody comprises a heavy chain having an aminoacid sequence having at least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 50, 54, 58, 60, 64, and 66; and a light chainhaving an amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 52, 56, 62 and 68.

In one embodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a heavy chain variable regionhaving an amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 42 and 46. In anotherembodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a light chain variable regionhaving an amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 44 and 48.

In another embodiment, the present invention provides a humanizedanti-PD-L1 antibody, wherein the antibody comprises a heavy chainvariable region having at least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to SEQ ID NO: 42 and a light chain variable regionhaving least 80% homology, at least 85% homology, at least 90% homology,at least 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toSEQ ID NO: 44. In another embodiment, the present invention provides ahumanized anti-PD-L1 antibody, wherein the antibody comprises a heavychain variable region having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to SEQ ID NO: 46 and a light chainvariable region having least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to SEQ ID NO: 48.

In one embodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a full heavy chain having anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 70, 72, 76, and 78. In another embodiment, thepresent invention provides a humanized anti-PD-L1 antibody, wherein theantibody comprises a full light chain having an amino acid sequencehaving at least 80% homology, at least 85% homology, at least 90%homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 74 and 80.

In one embodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 70 and a light chain according to SEQ ID NO: 74. In anotherembodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 72 and a light chain according to SEQ ID NO: 74. In anotherembodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 76 and a light chain according to SEQ ID NO: 80. In anotherembodiment, the present invention provides a humanized anti-PD-L1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 78 and a light chain according to SEQ ID NO: 80.

In one embodiment, the present invention provides anti-PD-L1 antibodiesor fragments thereof that bind to the same epitope on PD-L1 as any ofthe exemplary antibodies provided herein. In one embodiment, theantibodies or fragments thereof compete with any of the exemplaryantibodies provided herein for binding to PD-L1. Binding to PD-L1 may bemeasured by ELISA, flow cytometry, surface plasmon resonance (SPR)assay, or any other method known in the art.

In one embodiment, the present invention provides anti-PD-L1 antibodiesand fragments thereof that bind to PD-L1 with a binding affinity kD ofabout 10 nM to about 0.01 nM. In a further embodiment, the anti-PD-L1antibodies and fragments thereof provided herein bind to PD-L1 with abinding affinity kD of from about 10 nM to about 0.05 nM. In a furtherembodiment, the anti-PD-L1 antibodies and fragments thereof providedherein bind to PD-L1 with a binding affinity kD of from about 8 nM toabout 0.1 nM. In a further embodiment, the anti-PD-L1 antibodies andfragments thereof provided herein bind to PD-L1 with a binding affinitykD of from about 5 nM to about 0.2 nM. In another embodiment, theanti-PD-L1 antibodies and fragments thereof provided herein bind toPD-L1 with a binding affinity kD of about 10 nM or less. In a furtherembodiment, the anti-PD-L1 antibodies and fragments thereof providedherein bind to PD-L1 with a binding affinity kD of about 6 nM or less.In a further embodiment, the anti-PD-L1 antibodies and fragments thereofprovided herein bind to PD-L1 with a binding affinity kD of about 4 nMor less. In a further embodiment, the anti-PD-L1 antibodies andfragments thereof provided herein bind to PD-L1 with a binding affinitykD of about 2 nM or less. In a further embodiment, the anti-PD-L1antibodies and fragments thereof provided herein bind to PD-L1 with abinding affinity kD of about 1 nM or less. In a further embodiment, theanti-PD-L1 antibodies and fragments thereof provided herein bind toPD-L1 with a binding affinity kD of about 0.75 nM or less. In a furtherembodiment, the anti-PD-L1 antibodies and fragments thereof providedherein bind to PD-L1 with a binding affinity kD of about 0.5 nM or less.In a further embodiment, the anti-PD-L1 antibodies and fragments thereofprovided herein bind to PD-L1 with a binding affinity kD of about 0.25nM or less. In a further embodiment, the anti-PD-L1 antibodies andfragments thereof provided herein bind to PD-L1 with a binding affinitykD of about 0.2 nM or less. In a further embodiment, the anti-PD-L1antibodies and fragments thereof provided herein bind to PD-L1 with abinding affinity kD of about 0.15 nM or less. In a further embodiment,the anti-PD-L1 antibodies and fragments thereof provided herein bind toPD-L1 with a binding affinity kD of about 0.1 nM or less. In a furtherembodiment, the anti-PD-L1 antibodies and fragments thereof providedherein bind to PD-L1 with a binding affinity kD of about 0.075 nM orless. In a further embodiment, the anti-PD-L1 antibodies and fragmentsthereof provided herein bind to PD-L1 with a binding affinity kD ofabout 0.05 nM or less. In a further embodiment, the anti-PD-L1antibodies and fragments thereof provided herein bind to PD-L1 with abinding affinity kD of about 0.025 nM or less. In a further embodiment,the anti-PD-L1 antibodies and fragments thereof provided herein bind toPD-L1 with a binding affinity kD of about 0.02 nM or less. In a furtherembodiment, the anti-PD-L1 antibodies and fragments thereof providedherein bind to PD-L1 with a binding affinity kD of about 0.015 nM orless. In a further embodiment, the anti-PD-L1 antibodies and fragmentsthereof provided herein bind to PD-L1 with a binding affinity kD ofabout 0.01 nM or less. In one embodiment, the binding affinity kD of theanti-PD-L1 antibodies and fragments provided herein is measured byBiacore assay.

In one embodiment, the anti PD-L1 antibodies and fragments thereofprovided herein have a binding EC50 for PD-L1 of about 1 ng/mL to about2000 ng/mL. In a further embodiment, the anti PD-L1 antibodies andfragments thereof provided herein have a binding EC50 for PD-L1 of about1 ng/mL to about 1500 ng/mL. In a further embodiment, the anti PD-L1antibodies and fragments thereof provided herein have a binding EC50 forPD-L1 of about 1 ng/mL to about 1000 ng/mL. In a further embodiment, theanti PD-L1 antibodies and fragments thereof provided herein have abinding EC50 for PD-L1 of about 2 ng/mL to about 500 ng/mL. In a furtherembodiment, the anti PD-L1 antibodies and fragments thereof providedherein have a binding EC50 for PD-L1 of about 2 ng/mL to about 250ng/mL. In a further embodiment, the anti PD-L1 antibodies and fragmentsthereof provided herein have a binding EC50 for PD-L1 of about 5 ng/mLto about 200 ng/mL. In a further embodiment, the anti PD-L1 antibodiesand fragments thereof provided herein have a binding EC50 for PD-L1 ofabout 5 ng/mL to about 50 ng/mL. In one embodiment, the anti PD-L1antibodies and fragments thereof provided herein have a binding EC50 forPD-L1 of about 500 ng/mL or less, about 400 ng/mL or less, about 300ng/mL or less, about 250 ng/mL or less, about 200 ng/mL or less, about150 ng/mL or less, about 100 ng/mL or less, about 75 ng/mL or less,about 60 ng/mL or less, about 50 ng/mL or less, about 40 ng/mL or less,or about 30 ng/mL or less. In one embodiment, the EC50 of the anti-PD-L1antibodies and fragments provided herein is measured by ELISA or FACS.

In one embodiment, the anti PD-L1 antibodies and fragments thereofprovided herein inhibit PDL1/PD-1 binding with an IC50 of about of about1 ng/mL to about 1500 ng/mL. In a further embodiment, the anti PD-L1antibodies and fragments thereof provided herein inhibit PDL1/PD-1binding with an IC50 of about 2 ng/mL to about 1200 ng/mL. In a furtherembodiment, the anti PD-L1 antibodies and fragments thereof providedherein inhibit PDL1/PD-1 binding with an IC50 of about 5 ng/mL to about500 ng/mL. In a further embodiment, the anti PD-L1 antibodies andfragments thereof provided herein inhibit PDL1/PD-1 binding with an IC50of about 5 ng/mL to about 100 ng/mL. In a further embodiment, the antiPD-L1 antibodies and fragments thereof provided herein inhibit PDL1/PD-1binding with an IC50 of about 10 ng/mL to about 50 ng/mL. In oneembodiment, the anti PD-L1 antibodies and fragments thereof providedherein inhibit PDL1/PD-1 binding with an IC50 of about 1200 ng/mL orless, about 1000 ng/mL or less, about 800 ng/mL or less, about 400 ng/mLor less, about 300 ng/mL or less, about 250 ng/mL or less, about 200ng/mL or less, about 150 ng/mL or less, about 100 ng/mL or less, about75 ng/mL or less, about 60 ng/mL or less, about 50 ng/mL or less, about40 ng/mL or less, about 30 ng/mL or less, about 20 ng/mL or less, orabout 10 ng/mL or less. In one embodiment, the IC50 of the anti-PD-L1antibodies and fragments provided herein is measured by ELISA or FACS.

In one embodiment, the anti-PD-L1 antibody provided herein is ahumanized antibody having a heavy chain variable region amino acidsequence according to SEQ ID NO: 42 and a light chain variable regionamino acid according to SEQ ID NO: 44; or having a heavy chain variableregion amino acid sequence according to SEQ ID NO: 46 and a light chainvariable region amino acid sequence according to SEQ ID NO: 48; whereinthe anti-PD-L1 antibody has a PD-L1 binding EC50 of about 200 ng/ml orless or about 150 ng/mL or less or about 100 ng/mL or less or about 80ng/ml or less or about 60 ng/mL or less or about 50 ng/mL or less, asmeasured by ELISA or FACS. In another embodiment, the anti-PD-L1antibody provided herein is a humanized antibody having a heavy chainvariable region amino acid sequence according to SEQ ID NO: 42 and alight chain variable region amino acid according to SEQ ID NO: 44; orhaving a heavy chain variable region amino acid sequence according toSEQ ID NO: 46 and a light chain variable region amino acid sequenceaccording to SEQ ID NO: 48; wherein the anti-PD-L1 antibody has aPDL1/PD-1 blockage IC50 of about 1200 ng/mL or less, or about 1000 ng/mLor less, or about 800 ng/mL or less, or about 600 ng/mL or less, orabout 500 ng/mL or less, or about 400 ng/mL or less, or about 300 ng/mLor less, or about 200 ng/mL or less, or about 100 ng/mL or less, orabout 60 ng/mL or less, or about 30 ng/mL or less, or about 25 ng/mL orless, or about 20 ng/mL or less, or about 10 ng/mL or less, as measuredby ELISA or FACS. In another embodiment, the anti-PD-L1 antibodyprovided herein is a humanized antibody having a heavy chain variableregion amino acid sequence according to SEQ ID NO: 42 and a light chainvariable region amino acid according to SEQ ID NO: 44; or having a heavychain variable region amino acid sequence according to SEQ ID NO: 46 anda light chain variable region amino acid sequence according to SEQ IDNO: 48; wherein the anti-PD-L1 antibody has a binding affinity kD forPD-L1 of about 10 nM or less, or about 5 nM or less, or about 2 nM orless, or about 1 nM or less, or about 0.5 nM or less, or about 0.1 nM orless, or about 0.05 nM or less, as measured by Biacore assay. In oneembodiment, the humanized anti-PD-L1 antibody has a binding affinity kDfor PD-L1 of about 2 nM. In another embodiment, the humanized anti-PD-L1antibody has a binding affinity kD for PD-L1 of about 1 nM. In anotherembodiment, the humanized anti-PD-L1 antibody has a binding affinity kDfor PD-L1 of about 0.5 nM. In another embodiment, the humanizedanti-PD-L1 antibody has a binding affinity kD for PD-L1 of about 0.1 nM.

In one embodiment, the anti-PD-L1 antibodies and fragments thereofprovided herein bind to PD-L1, disrupting the PD-1/PD-L1 interaction andresulting in an increase in T cell activation. In a further embodiment,the antibodies and fragments thereof bind PD-L1 and result in anincrease in T cell proliferation and/or cytokine production. In a yetfurther embodiment, the antibodies and fragments thereof bind PD-L1 andresult in an increase of one or more cytokines selected from the groupconsisting of IL-2, IFNγ, TNF, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13,IL-17, and GM-CSF. Thus, in one aspect, the present invention providesmethods for modulating an immune response comprising contacting T cellsand antigen presenting cells with the anti-PD-L1 antibody or fragmentthereof. In one embodiment, the modulation of an immune response by theanti-PD-L1 antibodies and fragments provided herein may be measured in amixed lymphocyte (MLR) reaction. In one embodiment, the anti-PD-L1antibodies provided herein increase the level of cytokine productionfrom lymphocytes in an MLR. In a further embodiment, the anti-PD-L1antibodies increase the level of IL-2 production and/or IFNγ productionin an MLR. In a yet further embodiment, the anti-PD-L1 antibodiesincrease the level of IL-2 production and IFNγ production in an MLR. Inone embodiment, the anti-PD-L1 antibodies enhance memory T cellresponses. In a further embodiment, the anti-PD-L1 antibodies enhancememory T cell responses as measured by an increase in IFNγ productionfrom memory T cells.

In one embodiment, the anti-PD-L1 antibodies and fragments thereofprovided herein inhibit regulatory T cell function. In a furtherembodiment, the anti-PD-L1 antibodies and fragments thereof inhibit thesuppression of effector T cells by regulatory T cells. In anotherembodiment, the anti-PD-L1 antibodies and fragments thereof restore theeffector functions of T cells in the presence of regulatory T cells. Ina further embodiment, the anti-PD-L1 antibodies and fragments thereofrestore the ability of effector T cells to proliferate and/or producecytokines in the presence of regulatory T cells. Thus, in oneembodiment, the present invention provides a method for inhibiting thesuppressive effects of regulatory T cells in vitro or in a subject inneed thereof.

In one aspect, an isolated antibody or fragment thereof that binds toPD-L1 is provided, wherein the antibody is produced by a hybridomaselected from the group consisting of the hybridomas herein termed 13C5,5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, and 15F1. Thus, the presentinvention also encompasses the hybridomas 13C5, 5G9, 5G11, 8C6, 7B4,4D1, 4A8, 8H4, 8H3, and 15F1, as well as any hybridoma producing anantibody disclosed herein. The present invention also provides isolatedpolynucleotides encoding the antibodies and fragments thereof providedherein. Expression vectors comprising the isolated polynucleotides, andhost cells comprising such expression vectors, are also encompassed inthe invention.

In one embodiment, the present invention provides anti-PD-L1 antibodyimmunoconjugates. Thus, the present invention provides an antibody orfragment thereof that binds to PD-L1 and that is linked or conjugated toa therapeutic agent. Therapeutic agents that may be linked or conjugatedto the anti-PD-L1 antibody may include, but are not limited to,cytotoxic drugs, radioactive isotopes, immunomodulators, or antibodies.

In one aspect, the present invention provides compositions comprisingone or more anti-PD-L1 antibody or fragment thereof provided herein, anda pharmaceutically acceptable carrier.

In one aspect, the present invention provides methods for modulating animmune response in a subject, the method comprising administering to thesubject a therapeutically effective amount of an anti-PD-L1 antibody orfragment thereof provided herein. In one embodiment, the presentinvention provides methods for treating or preventing a disease ordisorder in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of an anti-PD-L1 antibody orfragment thereof provided herein.

In one embodiment, the present invention provides a method for enhancinganti-tumor responses in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ananti-PD-L1 antibody or fragment of the invention. In another embodiment,the present invention provides a method for reducing tumors orinhibiting the growth of tumor cells in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of an anti-PD-L1 antibody or fragment of the invention. Inanother embodiment, the present invention provides a method for treatingcancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of an anti-PD-L1 antibody orfragment of the invention. In a further embodiment, the cancer isselected from the group consisting of lymphoma, leukemia, melanoma,glioma, breast cancer, lung cancer, colon cancer, bone cancer, ovariancancer, bladder cancer, kidney cancer, liver cancer, stomach cancer,rectal cancer, testicular cancer, salivary cancer, thyroid cancer,thymic cancer, epithelial cancer, head or neck cancer, gastric cancer,pancreatic cancer, or a combination thereof.

In one embodiment, the present invention provides a method for treatingan infectious disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ananti-PD-L1 antibody or fragment of the invention. In a furtherembodiment, the infectious disease is selected from the group consistingof candidiasis, candidemia, aspergillosis, streptococcal pneumonia,streptococcal skin and oropharyngeal conditions, gram negative sepsis,tuberculosis, mononucleosis, influenza, respiratory illness caused byRespiratory Syncytial Virus, malaria, schistosomiasis, andtrypanosomiasis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a-d is set of graphs showing the binding of the murine hybridomaanti-PD-L1 antibodies to PD-L1 over a range of antibody concentrationsas measured by ELISA. Binding of hybridoma antibodies 8H3-mIgG (m8H3),15F1-mIgG (m15F1), 5G9-mIgG (m5G9), and 4A8-mIgG (m4A8) is shown in FIG.1a . Binding of hybridoma antibodies 5G11-mIgG (m5G11), 7B4-mIgG (m7B4),4D1-mIgG (m4D1), and 8H4-mIgG (m8H4) is shown in FIG. 1b . Binding ofhybridoma antibody 8C6-mIgG (m8C6) is shown in FIG. 1c . Binding ofhybridoma antibody 13C5-mIgG (m13C5) is shown in FIG. 1d . In each ofFIGS. 1a-1d , binding of mIgG1 is shown as a negative control.

FIG. 2a-c is set of graphs showing the binding of chimeric anti-PD-L1antibodies to PD-L1 over a range of concentrations as measured by ELISA.Binding of chimeric antibodies ch5G11-hIgG4 and ch5G11-hIgG1 is shown inFIG. 2a . Binding of chimeric antibodies ch13C5-hIgG4, ch13C5-hIgG1, andch8H4-hIgG4 is shown in FIG. 2b . Binding of chimeric antibodych8C6-hIgG4 is shown in FIG. 2c . In each of FIGS. 2a-2c , binding ofhIgG4 is shown as a negative control.

FIG. 3a-b is set of graphs showing the binding of humanized anti-PD-L1antibodies to PD-L1 over a range of antibody concentrations as measuredby ELISA. Binding of control hIgG4 and humanized antibodies hu5G11-hIgG1and hu5G11-hIgG4 is shown in FIG. 3a . Binding of control hIgG4 andhumanized antibodies hu13C5-hIgG1 and hu13C5-hIgG4 is shown in FIG. 3 b.

FIG. 4a-c is a set of graphs showing the blockage of the PD-1/PD-L1interaction by hybridoma anti-PD-L1 antibodies over a range of antibodyconcentrations as measured by ELISA. Blockage of PD-1/PD-L1 binding byhybridoma antibodies 13C5-mIgG (m13C5), 8C6-mIgG (m8C6), 5G9-mIgG(m5G9), and 4A8-mIgG (m4A8) as compared to control mIgG1 is shown inFIG. 4a . Blockage of PD-1/PD-L1 binding by hybridoma antibodies5G11-mIgG (m5G11), 7B4-mIgG (m7B4), 4D1-mIgG (m4D1), and 8H4-mIgG (m8H4)as compared to control mIgG1 is shown in FIG. 4b . Blockage ofPD-1/PD-L1 binding by hybridoma antibodies 8H3-mIgG (m8H3) and 15F1-mIgG(m15F1) as compared to control mIgG1 is shown in FIG. 4 c.

FIG. 5a-c is a set of graphs showing the blockage of the PD-1/PD-L1interaction by chimeric anti-PD-L1 antibodies over a range of antibodyconcentrations as measured by ELISA. Blockage of PD-1/PD-L1 binding bychimeric antibodies ch5G11 hIgG4 and ch5G11 hIgG1 as compared to controlhIgG4 is shown in FIG. 5a . Blockage of PD-1/PD-L1 binding by chimericantibody ch8C6-hIgG4 as compared to control hIgG4 is shown in FIG. 5b .Blockage of PD-1/PD-L1 binding by chimeric antibodies ch8H4-hIgG4,ch13C5-hIgG1, and ch13C5-hIgG4 as compared to control hIgG4 is shown inFIG. 5 c.

FIG. 6a-b is a set of graphs showing the blockage of the PD-1/PD-L1interaction by humanized anti-PD-L1 antibodies over a range of antibodyconcentrations as measured by ELISA. Blockage of PD-1/PD-L1 binding bycontrol hIgG4 and humanized antibodies 5G11-hIgG1 and 5G11-hIgG4 isshown in FIG. 6a . Blockage of PD-1/PD-L1 binding by control hIgG4 andhumanized antibodies 13C5-hIgG1 and 13C5-hIgG4 is shown in FIG. 6 b.

FIGS. 7a and 7b show the binding of the hybridoma anti-PD-L1 antibodiesto PD-L1 over a range of antibody concentrations as measured by FACS.Binding (as measured by the mean fluorescence intensity) of hybridomaantibodies 4A8, 15F1, 4D1, 13C5, 8H4, and 8H3 as compared to controlantibody mIgG1 is shown in FIG. 7a . Binding (as measured by the meanfluorescence intensity) of hybridoma antibodies 5G11, 8C6, 5G9, or 7B4as compared to control antibody mIgG1 is shown in FIG. 7 b.

FIG. 8 shows the binding of the chimeric anti-PD-L1 antibodies to PD-L1over a range of antibody concentrations as measured by FACS. Binding ofcontrol antibody hIgG4, and chimeric antibodies ch13C5-hIgG1,ch5G11-hIgG1, and ch5G11-hIgG4 are shown.

FIG. 9 shows the binding of humanized anti-PD-L1 antibodies to PD-L1over a range of antibody concentrations as measured by FACS. Binding ofcontrol antibody hIgG4 and humanized antibodies hu13C5-hIgG1,hu13C5-hIgG4, hu5G11-hIgG1, and hu5G11-hIgG4 are shown.

FIGS. 10a and 10b show the blockage of the PD-1/PD-L1 interaction byhybridoma anti-PD-L1 antibodies over a range of antibody concentrationsas measured by FACS. Blockage of PD-1/PD-L1 binding by control antibodymIgG1 and hybridoma antibodies m4D1, m5G11, m13C5, m7B4, and m8H4 isshown in FIG. 10a . Blockage of PD-1/PD-L1 binding by control antibodymIgG1 and hybridoma antibodies m4A8, m5G9, m8C6, m8H3, and m15F1 isshown in FIG. 10 b.

FIG. 11 shows the blockage of the PD-1/PD-L1 interaction over a range ofconcentrations of control antibody hIgG4 or chimeric anti-PD-L1antibodies ch8C6-hIgG4, ch5G11-hIgG1, ch5G11-hIgG4, ch13C5-hIgG1,ch13C5-hIgG4, or ch8H4-hIgG4, as measured by FACS.

FIG. 12 shows the blockage of the PD-1/PD-L1 interaction over a range ofconcentrations of control antibody hIgG4 or humanized antibodies hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1, or hu5G11-hIgG4, as measured byFACS.

FIG. 13a is a graph showing IL-2 (pg/mL) production in an MLR inresponse to different concentrations of hybridoma anti-PD-L1 antibodies.FIG. 13b is a graph showing IFNγ (pg/mL) production in an MLR inresponse to different concentrations of hybridoma anti-PD-L1 antibodies.For both FIGS. 13a and 13b , the antibodies tested were, from left toright, control mIgG1, m8C6, m4D1, m5G11, m7B4, m8H4, m5G9, m13C5, m8H3,and m15F1. T cell only and/or DC only wells were also included asnegative controls. As shown on the x-axis for both FIGS. 13a and 13b ,each antibody was tested at 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL,and 0.002 μg/mL.

FIG. 14a is a graph showing IL-2 (pg/mL) production in an MLR inresponse to different concentrations of chimeric anti-PD-L1 antibodies.FIG. 14b is a graph showing IFNγ (pg/mL) production in an MLR inresponse to different concentrations of chimeric anti-PD-L1 antibodies.For both FIGS. 14a and 14b , the antibodies tested were, from left toright, control hIgG4, chimeric 8C6-hIgG4, chimeric 8H4-hIgG4, chimeric5G11-hIgG4, and chimeric 13C5-hIgG1. As shown on the x-axis for bothFIGS. 14a and 14b , each antibody was tested at 20 μg/mL, 2 μg/mL, 0.2μg/mL, 0.02 μg/mL, and 0.002 μg/mL.

FIG. 15a is a graph showing IL-2 (pg/mL) production in an MLR inresponse to different concentrations of humanized anti-PD-L1 antibodies.FIG. 15b is a graph showing IFNγ (pg/mL) production in an MLR inresponse to different concentrations of humanized anti-PD-L1 antibodies.For both FIGS. 15a and 15b , the antibodies tested were, from left toright, control hIgG4, hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1, andhu5G11-hIgG4. As shown on the x-axis for both FIGS. 15a and 15b , eachantibody was tested at 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and0.002 μg/mL.

FIG. 16 shows the effects of chimeric (ch) or humanized (hu) anti-PD-L1antibodies on Treg-mediated inhibition of IFNγ production (pg/mL), in anallogeneic MLR with CD4+CD25+ Treg cells, CD4+CD25− T cells, anddendritic cells. The antibodies tested were, from left to right, controlhIgG4, ch13C5-hIgG1, ch13C5-hIgG4, hu13C5-hIgG1, hu13C5-hIgG4,ch5G11-hIgG1, ch5G11-hIgG4, hu5G11-hIgG1, and hu5G11-hIgG4.

FIG. 17 shows IFN-γ production (pg/mL) from T cells in response tocostimulation with autologous DCs and anti-CD3 antibody, in the presenceof humanized anti-PD-L1 antibody (hu13C5-hIgG1, hu13C5-hIgG4,hu5G11-hIgG1, or hu5G11-hIgG4), isotype control (hIgG4) antibody, or noantibody.

FIGS. 18a and 18b show the effect of humanized anti-PD-L1 antibodies onmemory T cell responses recalled by tetanus toxin, as measured by IFN-γproduction (pg/mL). Negative control hIgG4 or humanized antibodyhu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1, or hu5G11-hIgG4 were tested atthe following concentrations: 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL,and 0.002 μg/mL.

DETAILED DESCRIPTION

PD1/PDL1 interactions inhibit T cell receptor signaling by recruitingthe SHP1 and SHP2 phosphatases, which interfere with TCR signaling(Chemnitz et al. (2004) J. Immunol. 17:945-954). PD-L1 can not onlypromote tumor progression through inhibition of PD1-expressing immuneeffectors, but also modulate cell-mediated immunity in some infectiousdiseases (Mueller et al. (2010) J. Clin. Invest. 120:2508-2515).Furthermore, allogeneic effector T cell responses are susceptible toPD-1 pathway modulation in graft rejection (Lee et al. (2003) J.Immunol. 171:6929-6935). Therefore, the interaction of PD-1 with PD-L1exerts a vital and diverse range of immunoregulatory roles in T cellactivation, tolerance, and immune-mediated tissue damage. However, theinteraction can be reversed by blocking the local binding of PD-1 withPD-L1 (Iwai et al. (2002) Proc. Nat'l. Acad Sci. USA 99: 12293-7; Brownet al. (2003) J. Immunol. 170:1257-66).

PD-1 has been found to have a correlation with cancer growth anddevelopment due to its role in protecting tumor cells from efficientimmune destruction. Its ligand, PD-L1, has been revealed to havesignificant expression on a number of mouse and human tumors, which ispostulated to mediate immune evasion (Iwai, Y. et al., Proc. Natl. Acad.Sci. USA. 99: 12293-12297 (2002); Strome S. E. et al., Cancer Res.,63:6501-6505 (2003); Dong et al. (2002) Nat. Med. 8:787-9). In humans,expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 (ontumor cells) has been found in a number of primary tumor biopsies asassessed by immunohistochemistry. Such tissues include cancers of thelung, liver, ovary, cervix, skin, colon, glioma, bladder, breast,kidney, esophagus, stomach, oral squamous cell, urothelial cell, andpancreas as well as tumors of the head and neck (Brown J. A. et al., J.Immunol. 170: 1257-1266 (2003); Dong H. et al., Nat. Med. 8: 793-800(2002); Wintterle et al., Cancer Res. 63:7462-7467 (2003); Strome S. E.et al., Cancer Res., 63: 6501-6505 (2003); Thompson R. H. et al., CancerRes. 66: 3381-5(2006); Thompson et al., Clin. Cancer Res. 13:1757-61(2007); Nomi T. et al., Clin. Cancer Res. 13: 2151-7. (2007)).More strikingly, PD-1 ligand expression on tumor cells has beencorrelated to poor prognosis of cancer patients across multiple tumortypes (reviewed in OkaZaki and Honjo, Int. Immunol. 19: 813-824 (2007)).

While the interaction between PD-1 and PD-L1 results in a decrease intumor infiltrating lymphocytes, a decrease in T-cell receptor mediatedproliferation, and immune evasion by the cancerous cells (Dong et al.(2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol.Immunother. 54: 3 07-3 14; Konishi et al. (2004) Clin. Cancer Res.10:5094-100), blockade of the PD-1/PD-L1 interaction was accordinglyshown to enhance tumor-specific T-cell immunity and be helpful inclearance of tumor cells by the immune system. In a murine model ofaggressive pancreatic cancer, for example, Nomi T., et al. (Clin. CancerRes. 13: 2151-2157, 2007) demonstrated the therapeutic efficacy ofPD-1/PD-L1 blockade. Administration of either PD-1 or PD-L1 directedantibody significantly inhibited tumor growth. Antibody blockadeeffectively promoted tumor reactive CD8+ T cell infiltration into thetumor resulting in the up-regulation of anti-tumor effectors includingIFN-γ, granzyme B and perforin. Additionally, the authors showed thatPDL1/PD-1 blockade can be effectively combined with chemotherapy toyield a synergistic effect. In another study, using a model of squamouscell carcinoma in mice, antibody blockade of PD-1 or PD-L1 significantlyinhibited tumor growth (Tsushima F. et al., Oral Oncol. 42:268-274(2006)).

Furthermore, transfection of a murine mastocytoma line with PD-L1 led todecreased lysis of the tumor cells when co-cultured with atumor-specific CTL clone. Lysis was restored when anti-PD-L1 mAb wasadded (Iwai Y. et al., Proc. Natl. Acad. Sci. USA. 99: 12293-12297(2002)). In vivo, blocking the PD1/PD-L1 interaction was shown toincrease the efficacy of adoptive T cell transfer therapy in a mousetumor model (Strome S. E. et al., Cancer Res. 63:6501-6505 (2003)).Further evidence for the role of PD-1 in cancer treatment comes fromexperiments performed with PD-1 knockout mice. PD-L1 expressing myelomacells grew only in Wild-type animals (resulting in tumor growth andassociated animal death), but not in PD-1 deficient mice (Iwai Y., etal., Proc. Natl. Acad. Sci. USA. 99: 12293-12297(2002)). In humanstudies, R. M. Wong et al. (Int. Immunol. 19:1223-1234 (2007)) showedthat PD-1 blockade using a fully human anti-PD-1 antibody augmented theabsolute numbers of tumor-specific CD8+ T cells (CTLs) in ex vivostimulation assays using vaccine antigens and cells from vaccinatedindividuals. In a similar study, antibody blockade of PD-L1 resulted inenhanced cytolytic activity of tumor-associated antigen-specificcytotoxic T cells and increased cytokine production by tumor specific THcells (Blank C. et al., Int. J. Cancer 119: 317-327 (2006)). The sameauthors showed that PD-L1 blockade augments tumor-specific T cellresponses in vitro when used in combination with anti-CTLA-4 blockade.Overall, the PD-1/PD-L1 pathway is a target for the development ofantibody therapeutics for cancer treatment. Anti-PD-L1 antibodies mayalso be useful in chronic viral infection. Memory CD8+ T cells generatedafter an acute viral infection are highly functional and constitute animportant component of protective immunity. In contrast, chronicinfections are often characterized by varying degrees of functionalimpairment (exhaustion) of virus-specific T-cell responses, and thisdefect is a principal reason for the inability of the host to eliminatethe persisting pathogen. Although functional effector T cells areinitially generated during the early stages of infection, they graduallylose function during the course of a chronic infection. Barber et al.(Barber et al., Nature 439: 682-687 (2006)) showed that mice infectedwith a laboratory strain of LCMV developed chronic infection resultingin high levels of virus in the blood and other tissues. These miceinitially developed a robust T cell response, but eventually succumbedto the infection upon T cell exhaustion. The authors found that thedecline in number and function of the effector T cells in chronicallyinfected mice could be reversed by injecting an antibody that blockedthe interaction between PD-1 and PD-L1.

In one aspect, the present invention provides antibodies or antigenbinding fragments thereof that bind to programmed death ligand 1(PD-L1). In one embodiment, the antibodies or fragments thereof bind tohuman PD-L1. In another embodiment, the antibodies or fragments thereofbind to human and to cynomolgous PD-L1. In another embodiment, theantibodies or fragments thereof block the interaction of PD-L1 with itsreceptor PD-1 on T cells. In one aspect, the present invention providesmethods of making and using the anti-PD-L1 antibodies or fragmentsthereof, and compositions comprising anti-PD-L1 antibodies or fragmentsthereof, including pharmaceutical compositions.

As used herein, the term “antibody” refers to a binding protein havingat least one antigen binding domain. The antibodies and fragmentsthereof of the present invention may be whole antibodies or any fragmentthereof. Thus, the antibodies and fragments of the invention includemonoclonal antibodies or fragments thereof and antibody variants orfragments thereof, as well as immunoconjugates. Examples of antibodyfragments include Fab fragments, Fab′ fragments, F(ab)′ fragments, Fvfragments, isolated CDR regions, single chain Fv molecules (scFv), andother antibody fragments known in the art. Antibodies and fragmentsthereof may also include recombinant polypeptides, fusion proteins, andbi-specific antibodies. The anti-PD-L1 antibodies and fragments thereofdisclosed herein may be of an IgG1, IgG2, IgG3, or IgG4 isotype. Theterm “isotype” refers to the antibody class encoded by the heavy chainconstant region genes. In one embodiment, the anti-PD-L1 antibodies andfragments thereof disclosed herein are of an IgG1 or an IgG4 isotype.The PD-L1 antibodies and fragments thereof of the present invention maybe derived from any species including, but not limited to, mouse, rat,rabbit, primate, llama, and human. The PD-L1 antibodies and fragmentsthereof may be chimeric, humanized, or fully human antibodies. In oneembodiment, the anti-PD-L1 antibodies are antibodies produced by ahybridoma cell line derived from a mouse. Thus, in one embodiment, theanti-PD-L1 antibodies are murine antibodies. In another embodiment, theanti-PD-L1 antibodies are chimeric antibodies. In a further embodiment,the chimeric antibodies are mouse-human chimeric antibodies. In anotherembodiment, the antibodies are humanized antibodies. In a furtherembodiment, the antibodies are derived from murine antibodies and arehumanized.

A “chimeric antibody” is an antibody having at least a portion of theheavy chain variable region and at least a portion of the light chainvariable region derived from one species; and at least a portion of aconstant region derived from another species. For example, in oneembodiment, a chimeric antibody may comprise murine variable regions anda human constant region.

A “humanized antibody” is an antibody containing complementaritydetermining regions (CDRs) that are derived from a non-human antibody;and framework regions as well as constant regions that are derived froma human antibody. For example, the anti-PD-L1 antibodies provided hereinmay comprise CDRs derived from one or more murine antibodies and humanframework and constant regions. Thus, in one embodiment, the humanizedantibody provided herein binds to the same epitope on PD-L1 as themurine antibody from which the antibody's CDRs are derived. Exemplaryhumanized antibodies are provided herein. Additional anti-PD-L1antibodies comprising the heavy and light chain CDRs provided herein, orvariants thereof, may be generated using any human framework sequence,and are also encompassed in the present invention. In one embodiment,framework sequences suitable for use in the present invention includethose framework sequences that are structurally similar to the frameworksequences provided herein. Further modifications in the frameworkregions may be made to improve the properties of the antibodies providedherein. Such further framework modifications may include chemicalmodifications; point mutations to reduce immunogenicity or remove T cellepitopes; or back mutation to the residue in the original germlinesequence. In some embodiments, such modifications include thosecorresponding to the mutations exemplified herein, includingbackmutations to the germline sequence. For example, in one embodiment,one or more amino acids in the human framework regions of the VH and/orVL of the humanized antibodies provided herein are back mutated to thecorresponding amino acid in the parent murine antibody. As an example,as for VH and VL of humanized 5G11 and humanized 13C5, several sites offramework amino acid of the aforementioned template human antibody wereback mutated to the corresponding amino acid sequences in mouse 5G11 and13C5 antibodies. In one embodiment, the amino acid at positions 53and/or 60 and/or 67 of the light chain variable region is back mutatedto the corresponding amino acid found at that position in the mouse 5G11or 13C5 light chain variable region. In another embodiment, the aminoacid at positions 24 and/or 28 and/or 30 and/or 49 and/or 73 and/or 83and/or 94 of the heavy chain variable region is back mutated to thecorresponding amino acid found at that position in the mouse 5G11 or13C5 heavy chain variable region. In one embodiment, the humanized 5G11antibody comprises a light chain variable region wherein the amino acidat position 60 is mutated from Ser (S) to Asp (D) and the amino acid atposition 67 is mutated from Ser (S) to Tyr (Y); and a heavy chainvariable region wherein the amino acid at position 24 is mutated fromPhe (F) to Val (V), the amino acid at position 49 is mutated from Ala(A) to Gly (G), the amino acid at position 73 is mutated from Thr (T) toAsn (N), and the amino acid at position 83 is mutated from Thr (T) toAsn (N). In one embodiment, the humanized 13C5 antibody comprises alight chain variable region wherein the amino acid at position 53 ismutated from Tyr (Y) to Lys (K); and a heavy chain variable regionwherein the amino acid at position 28 is mutated from Thr (T) to Ile(I), the amino acid at position 30 is mutated from Ser (S) to Arg (R),the amino acid at position 49 is mutated from Ser (S) to Ala (A), andthe amino acid at position 94 is mutated from Tyr (Y) to Asp (D).Additional or alternate back mutations may be made in the frameworkregions of the humanized antibodies provided herein in order to improvethe properties of the antibodies. The present invention also encompasseshumanized antibodies that bind to PD-L1 and comprise frameworkmodifications corresponding to the exemplary modifications describedherein with respect to any suitable framework sequence, as well as otherframework modifications that otherwise improve the properties of theantibodies.

As used herein, the term “derived” when used to refer to a molecule orpolypeptide relative to a reference antibody or other binding protein,means a molecule or polypeptide that is capable of binding withspecificity to the same epitope as the reference antibody or otherbinding protein.

The antibodies and antigen-binding fragments thereof disclosed hereinare specific for PD-L1. In one embodiment, the antibodies and fragmentsthereof are specific for human PD-L1. In one embodiment, the antibodiesand fragments provided herein bind to human or primate PD-L1 but not toPD-L1 from any other mammal. In a further embodiment, the antibodies andfragments thereof do not bind to mouse PD-L1. The terms “human PD-L1,”“hPD-L1”, and “huPD-L1” and the like are used interchangeably herein andrefer to human PD-L1 and variants or isoforms of human PD-L1. By“specific for” is meant that the antibodies and fragments thereof bindPD-L1 with greater affinity than any other target. As used herein, theterm “EC50” refers to the effective concentration, 50% maximal responseof the antibody. As used herein, the term “IC50” refers to theinhibitory concentration, 50% maximal response of the antibody. BothEC50 and IC50 may be measured by ELISA or FACS analysis, or any othermethod known in the art.

In one embodiment, the anti-PD1 antibodies and fragments or variantsthereof have a binding affinity (KD) for PD-L1 in the range of about0.001 nM to about 100 nM, about 0.002 nM to about 50 nM, about 0.005 nMto about 5 nM, about 0.01 nM to about 1 nM, or about 0.05 nM to about0.1 nM. In one embodiment, the antibodies and fragments thereof have abinding affinity (KD) for PD-L1 of about 50 nM or less, about 25 nM orless, about 20 nM or less, about 15 nM or less, about 10 nM or less,about 8 nM or less, about 6 nM or less, about 5 nM or less, about 4 nMor less, about 3 nM or less, about 2 nM or less, about 1 nM or less,about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nMor less, about 0.2 nM or less, about 0.1 nM or less, about 0.09 nM orless, about 0.08 nM or less, about 0.07 nM or less, about 0.06 nM orless, about 0.05 nM or less, about 0.04 nM or less, about 0.03 nM orless, about 0.02 nM or less, about 0.01 nM or less, about 0.009 nM orless, about 0.008 nM or less, about 0.007 nM or less, about 0.006 nM orless, about 0.005 nM or less, about 0.004 nM or less, about 0.003 nM orless, about 0.002 nM or less, or about 0.001 nM or less. In oneembodiment, the antibodies and fragments thereof have a binding affinity(KD) for PD-L1 of about 10 nM, about 9 nM, about 8 nM, about 7 nM, about6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.09 nM, about0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM, about 0.04 nM,about 0.03 nM, about 0.02 nM, about 0.01 nM, about 0.009 nM, about 0.008nM, about 0.007 nM, about 0.006 nM, about 0.005 nM, about 0.004 nM,about 0.003 nM, about 0.002 nM, or about 0.001 nM.

In one embodiment, the antibodies and fragments provided herein comprisea light chain and a heavy chain, each of which comprises three CDRregions. Exemplary heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3)for PD-L1 antibodies of the invention are provided below in Table 1.Exemplary light chain CDR sequences (LCDR1, LCDR2, and LCDR3) for PD-L1antibodies of the invention are provided below in Table 2. Exemplaryvariable regions and full length heavy and light chain sequences forPD-L1 antibodies of the invention are provided below in Table 3.

TABLE 1 Heavy Chain CDR Sequences Name HCDR SEQ ID NO Sequence 13C5 1 81 SYGMS 2  82 SISSGGSTYYPDSVKG 3  83 GYDSGFAY 5G9 1  87 SYGMS 2  88SISSGGTTYYPDSVKG 3  89 GYDSGFAY 5G11 1  93 TYGVH 2  94 VIWRGVTTDYNAAFMS3  95 LGFYAMDY 8C6 1  99 SYGVH 2 100 VIWSGGVTDYNAAFIS 3 101 LGFYAMDY 7B41 105 TYWMH 2 106 QINPDSTTINYAPSLKD 3 107 PGDYGYDFDC 4D1 1 111 SGYWN 2112 YISYSGSTYYNPSLKS 3 113 SLLWFSTGFAY 4A8 1 117 SYGVH 2 118VIWSGGITDYNAAFKS 3 119 LGFYAMDY 8H4 1 123 SYGMS 2 124 SISSGGTTYYLGSVQG 3125 GYDAGFAY 8H3 1 129 SGYWT 2 130 YISYTGSTYYNPSLKS 3 131 QRDWLGFAY 15F11 135 SYGMS 2 136 SISSGGSIYYPDSVKG 3 137 GYDAGFAF

TABLE 2 Light chain CDR Sequences Name LCDR SEQ ID NO Sequence 13C5 1 84 ASQSVSTSSSSFMH 2  85 YASNLES 3  86 QHSWEIPYT 5G9 1  90RASQSVSTSSSSYMH 2  91 YASNLES 3  92 QHSWEIPYT 5G11 1  96 KASQSVSNDVA 2 97 YAANRYT 3  98 QQDYTSPYT 8C6 1 102 KASQSVSNDVG 2 103 YASNRYS 3 104QQDYTSPYT 7B4 1 108 RSSQIIVHSNANTYLE 2 109 KVSNRFS 3 110 FQGSHVPYT 4D1 1114 SASSSVSSSYLY 2 115 NTSNLAS 3 116 HQWRSYPPT 4A8 1 120 SANSSVSYMH 2121 DTSKLAS 3 122 QQWSSNPWT 8H4 1 126 RASQSVSTSSYSYMH 2 127 YASNLES 3128 QNSWEIPYT 8H3 1 132 KSSQSLLYSSNQKNSLA 2 133 WASNRES 3 134 QQYYSYPLT15F1 1 138 RASQSVSTSSYSYVH 2 139 YASNLES 3 140 QHSWEIPYT

TABLE 3Heavy chain and light chain variable region and full length heavy and lightchain amino acid sequences SEQ ID Name Region NO Sequence 13C5 Heavy  2EVKLVESGGGLVKPGGSLKLSCAASGFIFRSYGMSWVRQTPE murine chainKRLEWVASISSGGSTYYPDSVKGRFTISRDNAR variableNILYLQMSSLRSEDTAMYDCARGYDSGFAYWGQGTLVTVSE 13C5 Light  4DIVLTQSPASLAVSLGQRATISCRASQSVSTSSSSFMHWYQQK murine chainPGQPPKLLIKYASNLESGVPARFSGSGSGTDFT variableLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKR 5G9 Heavy  6EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWVRQTP murine chainEKRLEWVASISSGGTTYYPDSVKGRFIISRDNARNILYLQMSS variableLRSEDTAMYYCAKGYDSGFAYWGQGTLVIVSA 5G9 Light  8DIVLTQSPPSLAVSLGQRATISCRASQSVSTSSSSYMHWYQQK murine chainPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDT variableATYYCQHSWEIPYTFGGGTKLEIK 5G11 Heavy 10QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPG murine chainKGLEWLGVIWRGVTTDYNAAFMSRLTITKDNSKSQVFFKMN variableSLQANDTAIYYCARLGFYAMDYWGQGTSVTVSS 5G11 Light 12SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPG murine chainQSPKLLIYYAANRYTGVPDRFTGSGYGTDFTFTISIVQAEDLA variableVYFCQQDYTSPYTFGGGTKLEIK 8C6 murine Heavy 14QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPG chainKGLEWLGVIWSGGVTDYNAAFISRLSISKDNSKSQVFFKMNS variableLQANDTAIYYCARLGFYAMDYWGQGTSVTVSS 8C6 murine Light 16SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVGWYQQKPG chainQSPKLLIYYASNRYSGVPDRFTGSGYGTDFTFTISTVQAEDLA variableVYFCQQDYTSPYTFGGGTKLEIK 7B4 murine Heavy 18EVKLFESGGGLVQPGGSLKLSCVASGFDFSTYWMHWVRQAP chainGQGLEWIGQINPDSTTINYAPSLKDRFIISRDNAKNTLFLQMS variableKVRSEDTALYYCAKPGDYGYDFDCWGQGTTLTVSS 7B4 murine Light 20DVLMTQTPLYLPVSLGDQASISCRSSQIIVHSNANTYLEWFLQ chainKPGQSPKWYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE variableDLGVYYCFQGSHVPYTFGGGTKLEIK 4D1 murine Heavy 22EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGN chainKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVT variableTEDTATYYCARSLLWFSTGFAYWGQGTLVTVSA 4D1 murine Light 24QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWNQQKPGS chainSPKVWIYNTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAAS variableYFCHQWRSYPPTLGAGTKLELK 4A8 murine Heavy 26QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPG chainKGLEWLGVIWSGGITDYNAAFKSRLSISKDNSKSQVFFKMNS variableLQANDTAIYFCARLGFYAMDYWGQGTSVTVSS 4A8 murine Light 28QIVLTQSPAIMSASPGEKVTMTCSANSSVSYMHWYQQKSGTS chainPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMGAEDAAT variableYYCQQWSSNPWTFGGGTKLEIK 8H4 murine Heavy 30EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWARQIPE chainKRLEWVASISSGGTTYYLGSVQGRFTISRDNARNILYLQMSSL variableRSEDTAMYYCARGYDAGFAYWGQGTLVSVSE 8H4 murine Light 32DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQ chainKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEE variableDTATYYCQNSWEIPYTFGGGTKLEIK 8H3 murine Heavy 34EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWTWIRKFPGN chainKLEYMGYISYTGSTYYNPSLKSRISISRDTSKSQYYLQLNSVT variableTEDTATYYCARQRDWLGFAYWGQGTLVTVSA 8H3 murine Light 36DIVMTQTPSSLAVSLGEKVTMSCKSSQSLLYSSNQKNSLAWY chainQQKPGQSPKLLIYWASNRESGVPDRFTGSSSGTDFTLTISSVK variableAEDLAVYYCQQYYSYPLTFGAGTKLELK 15F1 murine Heavy 38EEKLVESGGGLVKPGGSLKLSCAASGFSFSSYGMSWVRQTPE chainKRLEWVASISSGGSIYYPDSVKGRFTISRDNARNILYLQMSSL variableRSEDTAMYYCARGYDAGFAFWGQGTLVTASA 15F1 murine Light 40DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYVHWYQQ chainKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEE variableDTATYYCQHSWEIPYTFGGGTKLEIK 5G11 Heavy 42QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGK humanized chainALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMN variableNMDPVDTATYYCARLGFYAMDYWGQGTLVTVSS 5G11 Light 44DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGK humanized chainAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIAT variableYFCQQDYTSPYTFGQGTKLEIK 13C5 Heavy 46EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAP humanized chainGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMN variableSLRAEDTAVYDCARGYDSGFAYWGQGTLVTVSS 13C5 Light 48DIVLTQSPASLAVSPGQRATITCRASQSVSTSSSSFMHWYQQK humanized chainPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTINPVEAND variableTANYYCQHSWEIPYTFGQGTKLEIK Chimeric Full 50QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPG 8C6-IgG4 lengthKGLEWLGVIWSGGVTDYNAAFISRLSISKDNSKSQVFFKMNS (F234A/ heavyLQANDTAIYYCARLGFYAMDYWGQGTSVTVSSASTKGPSVF L235A) chainPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH (IgG4)TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGChimeric Full 52 SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVGWYQQKPG 8C6 lengthQSPKLLIYYASNRYSGVPDRFTGSGYGTDFTFTISTVQAEDLA lightVYFCQQDYTSPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS chainGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC Chimeric Full 54EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWARQIPE 8H4-IgG4 lengthKRLEWVASISSGGTTYYLGSVQGRFTISRDNARNILYLQMSSL (F234A/ heavyRSEDTAMYYCARGYDAGFAYWGQGTLVSVSSASTKGPSVFP L235A) chainLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT (IgG4)FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGChimeric Full 56 DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQ 8H4 lengthKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEE lightDTATYYCQNSWEIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ chainLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Chimeric Full 58QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPG 5G11-IgG1 lengthKGLEWLGVIWRGVTTDYNAAFMSRLTITKDNSKSQVFFKMN (D265A) heavySLQANDTAIYYCARLGFYAMDYWGQGTSVTVSSASTKGPSV chainFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV (IgG1)HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKChimeric Full 60 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPG 5G11-IgG4length KGLEWLGVIWRGVTTDYNAAFMSRLTITKDNSKSQVFFKMN (F234A/ heavySLQANDTAIYYCARLGFYAMDYWGQGTSVTVSSASTKGPSV L235A) chainFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV (IgG4)HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGChimeric Full 62 SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPG 5G11 lengthQSPKLLIYYAANRYTGVPDRFTGSGYGTDFTFTISIVQAEDLA lightVYFCQQDYTSPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS chainGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC Chimeric Full 64EVKLVESGGGLVKPGGSLKLSCAASGFIFRSYGMSWVRQTPE 13C5-IgG1 lengthKRLEWVASISSGGSTYYPDSVKGRFTISRDNARNILYLQMSSL (D265A) heavyRSEDTAMYDCARGYDSGFAYWGQGTLVTVSSASTKGPSVFP chainLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT (IgG1)FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKChimeric Full 66 EVKLVESGGGLVKPGGSLKLSCAASGFIFRSYGMSWVRQTPE 13C5-IgG4length KRLEWVASISSGGSTYYPDSVKGRFTISRDNARNILYLQMSSL (F234A/ heavyRSEDTAMYDCARGYDSGFAYWGQGTLVTVSSASTKGPSVFP L235A) chainLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT (IgG4)FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGChimeric Full 68 DIVLTQSPASLAVSLGQRATISCRASQSVSTSSSSFMHWYQQK 13C5 lengthPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDT lightATYYCQHSWEIPYTFGGGTKLEIKRTRTVAAPSVFIFPPSDEQ chainLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Humanized Full 70QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGK 5G11-IgG1 lengthALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMN (D265A) heavyNMDPVDTATYYCARLGFYAMDYWGQGTLVTVSSASTKGPS chainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG (IgG1)VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKHumanized Full 72 QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGK 5G11-IgG4length ALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMN (F234A/ heavyNMDPVDTATYYCARLGFYAMDYWGQGTLVTVSSASTKGPS L235A) chainVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG (IgG4)VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGHumanized Full 74 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGK 5G11 lengthAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIAT lightYFCQQDYTSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSG chainTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC Humanized Full 76EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAP 13C5-IgG1 lengthGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMN (D265A) heavySLRAEDTAVYDCARGYDSGFAYWGQGTLVTVSSASTKGPSV chainFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV (IgG1)HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKHumanized Full 78 EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAP 13C5-IgG4length GKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMN (F234A/ heavySLRAEDTAVYDCARGYDSGFAYWGQGTLVTVSSASTKGPSV L235A) chainFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV (IgG4)HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGHumanized Full 80 DIVLTQSPASLAVSPGQRATITCRASQSVSTSSSSFMHWYQQK 13C5length PGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTINPVEAND lightTANYYCQHSWEIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL chainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

In one embodiment, the invention provides anti-PD-L1 antibodies thatcomprise the light chain CDRs and heavy chain CDRs of antibodies 13C5,5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, and/or 15F1. The person ofskill in the art will understand that the heavy and light chain CDRs ofthe antibodies provided herein may be independently selected, or mixedand matched, to form an antibody or binding fragment thereof comprisingany heavy chain CDR1, CDR2, and CDR3; and any light chain CDR1, CDR2,and CDR3 from the antibodies provided herein. Thus, the inventionprovides anti-PD-L1 antibodies that comprise a heavy chain CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 81, 87, 93, 99, 105, 111, 117, 123, 129, and 135; a heavychain CDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 82, 88, 94, 100, 106, 112, 118, 124, 130, and136; a heavy chain CDR3 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 83, 89, 95, 101, 107, 113, 119, 125,131, and 137; a light chain CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 84, 90, 96, 102, 108,114, 120, 126, 132, and 138; a light chain CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 85, 91, 97,103, 109, 115, 121, 127, 133, and 139; and a light chain CDR3 comprisingan amino acid sequence selected from the group consisting of SEQ IDNOs:86, 92, 98, 104, 110, 116, 122, 128, 134, and 140. In oneembodiment, the present invention provides anti-PD-L1 antibodiescomprising heavy and light chain CDR regions comprising amino acidsequences having at least 75%, at least 80%, at least at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% homology to the correspondinglight or heavy chain CDR1, CDR2, or CDR3 provided herein. In oneembodiment, the present invention provides anti-PD-L1 antibodiescomprising heavy and light chain CDR regions comprising amino acidsequences having 1, 2, 3, 4, 5, or 6 amino acid substitutions,deletions, or insertions relative to the corresponding light or heavychain CDR1, CDR2, or CDR3 provided herein.

In one embodiment, the invention provides anti-PD-L1 antibodies thatcomprise a variable heavy chain of an antibody selected from the groupconsisting of 13C5, 5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, and/or 15F1and a variable light chain of an antibody selected from the groupconsisting of 13C5, 5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, and/or15F1. In one embodiment, the antibodies and fragments provided hereincomprise a heavy chain variable region comprising an amino acid sequencethat is at least 75%, at least 80%, at least at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% homology to a heavy chain variable regionselected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 30, 34, 38, 42, and 46. In one embodiment, the antibodies andfragments provided herein comprise a heavy chain variable regioncomprising an amino acid sequence according to SEQ ID NO: 2, 6, 10, 14,18, 22, 26, 30, 34, 38, 42, 46, or a variant thereof, wherein thevariant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions or deletions, or a combination thereof. In a furtherembodiment, the amino acid substitutions are conservative substitutions.

In one embodiment, the antibodies and fragments provided herein comprisea light chain variable region comprising an amino acid sequence that isat least 75%, at least 80%, at least at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% homology to a light chain variable regionselected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24,28, 32, 36, 40, 44, or 48. In one embodiment, the antibodies andfragments provided herein comprise a light chain variable regioncomprising an amino acid sequence according to SEQ ID NO: 4, 8, 12, 16,20, 24, 28, 32, 36, 40, 44, 48, or a variant thereof, wherein thevariant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acidsubstitutions, insertions, or deletions, or a combination thereof. In afurther embodiment, the amino acid substitutions are conservativesubstitutions.

The anti-PD-L1 antibodies disclosed herein having one or more amino acidsubstitution, insertion, deletion, or combination thereof in the CDR orvariable light or heavy chain region retain the biological activity ofthe corresponding anti-PD-L1 antibody that does not have an amino acidsubstitution, insertion, or deletion. Thus, the variant anti-PD-L1antibodies provided herein retain binding to PD-L1. Percent homology, asused herein, refers to the number of identical amino acid sequencesshared by two reference sequences, divided by the total number of aminoacid positions, multiplied by 100.

In some embodiments, the anti-PD-L1 antibodies provided herein compriseconservative amino acid substitutions. The person of skill in the artwill recognize that a conservative amino acid substitution is asubstitution of one amino acid with another amino acid that has asimilar structural or chemical properties, such as, for example, asimilar side chain. Exemplary conservative substitutions are describedin the art, for example, in Watson et al., Molecular Biology of theGene, The Bengamin/Cummings Publication Company, 4^(th) Ed. (1987).

The skilled person will understand that the variable light and variableheavy chains may be independently selected, or mixed and matched, fromthe antibodies provided herein. Thus, the present invention providesanti-PD-L1 antibodies comprising a heavy chain variable region having atleast 80% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and46; and a light chain variable region having at least 80% homology to anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48.

In one embodiment, the present invention provides antibodies that bindto the same epitope as any one of the exemplary antibodies disclosedherein. Thus, in one embodiment, the present invention providesantibodies that compete for binding to PD-L1 with the exemplaryantibodies provided herein.

The anti-PD-L1 antibodies and fragments thereof provided herein mayfurther comprise Fc region modifications to alter effector functions. Fcmodifications may be amino acid insertions, deletions, or substitutions,or may be chemical modifications. For example, Fc region modificationsmay be made to increase or decrease complement binding, to increase ordecrease antibody-dependent cellular cytoxicity, or to increase ordecrease the half life of the antibody. Some Fc modifications increaseor decrease the affinity of the antibody for an Fcγ receptor such asFcγRI, FcγRII, FcγRIII, or FcRn. Various Fc modifications have beendescribed in the art, for example, in Shields et al., J Biol. Chem 276;6591 (2001); Tai et al. Blood 119; 2074 (2012); Spiekermann et al. JExp. Med 196; 303 (2002); Moore et al. mAbs 2:2; 181 (2010);Medzihradsky Methods in Molecular Biology 446; 293 (2008); Mannan et al.Drug Metabolism and Disposition 35; 86 (2007); and Idusogie et al. JImmunol 164; 4178 (2000). In some embodiments, Fc region glycosylationpatters are altered. In other embodiments, the Fc region is modified bypegylation (e.g., by reacting the antibody or fragment thereof withpolyethylene glycol (PEG).

In one embodiment, the antibodies or fragments thereof provided hereinare immunoconjugates comprising an anti-PD-L1 antibody or fragmentthereof and further comprising an agent selected from the groupincluding an additional therapeutic agent, a cytotoxic agent, animmunoadhesion molecule, and an imaging agent. In some embodiments, theimaging agent is selected from the group consisting of a radiolabel, anenzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, and biotin. In some embodiments, the imagingagent is a radiolabel selected from the group consisting of: 3H, 14C,³⁵S, ⁶²Cu, ⁶⁴Cu, ⁸⁹Zr, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,¹⁵³Sm. In some embodiments, the therapeutic agent or cytotoxic agent isselected from the group including a chemotherapeutic agent, animmunosuppressive agent, an immuno-stimulatory agent, ananti-metabolite, an alkylating agent, an antibiotic, a growth factor, acytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, a toxin, and an apoptotic agent. In some embodiments, thebinding protein is conjugated directly to the agent. In otherembodiments, the binding protein is conjugated to the agent via alinker. Suitable linkers include, but are not limited to, amino acid andpolypeptide linkers disclosed herein. Linkers may be cleavable ornon-cleavable.

In one embodiment, the present invention provides bispecific ormultispecific antibodies specific for PD-L1 and at least one otherantigen or epitope. The anti-PD-L1 antibodies and fragments thereofprovided herein may be tested for binding to PD-L1 using the bindingassays provided herein, or any other binding assay known in the art.

Unless otherwise stated, the practice of the present invention employsconventional molecular biology, cell biology, biochemistry, andimmunology techniques that are well known in the art and described, forexample, in Methods in Molecular Biology, Humana Press; MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989),Current Protocols in Immunology (J. E. Coligan et al., eds., 1991);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); Phagedisplay: a laboratory manual (C. Barbas III et al, Cold Spring HarborLaboratory Press, 2001); and Using antibodies: a laboratory manual (E.Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999).

In one aspect the present invention provides methods for treating asubject for a disease or condition responsive to enhancing, stimulating,or eliciting an immune response. As used herein, the terms “treatment”or “treating” refers to both therapeutic treatment and prophylactic orpreventive measures. Subjects in need of treatment include thosesubjects that already have the disease or condition, as well as thosethat may develop the disease or condition and in whom the object is toprevent, delay, or diminish the disease or condition. As used herein,the term “subject” denotes a mammal, such as a rodent, a feline, acanine, and a primate. Preferably, a subject according to the inventionis a human.

The term “therapeutically effective amount,” as used herein, refers tothe amount of a compound or composition that is necessary to provide atherapeutic and/or preventative benefit to the subject.

In one aspect, the antibodies and antigen binding fragments thereof areuseful in the treatment of solid or non-solid tumors. Thus, in oneaspect, the present invention provides methods for treatment of cancer.“Cancer” as used herein refers to the physiological condition in mammalsthat is typically characterized by unregulated cell growth. Examples ofcancer include but are not limited to carcinoma, lymphoma, blastoma,sarcoma (including liposarcoma, osteogenic sarcoma, angiosarcoma,endotheliosarcoma, leiomyosarcoma, chordoma, lymphangiosarcoma,lymphangioendotheliosarcoma, rhabdomyosarcoma, fibrosarcoma,myxosarcoma, chondrosarcoma), neuroendocrine tumors, mesothelioma,synovioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemiaor lymphoid malignancies. More particular examples of such cancersinclude squamous cell cancer (e.g. epithelial squamous cell cancer),Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, smalllymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides,mantle cell lymphoma, follicular lymphoma, diffuse large B-celllymphoma, marginal zone lymphoma, hairy cell leukemia andlymphoplasmacytic leukemia), tumors of lymphocyte precursor cells,including B-cell acute lymphoblastic leukemia/lymphoma, and T-cell acutelymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T and NKcells, including peripheral T-cell leukemias, adult T-cellleukemia/T-cell lymphomas and large granular lymphocytic leukemia,Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia, B-cell acute lymphoblastic leukemia/lymphoma, T-cell acutelymphoblastic leukemia/lymphoma, lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, small cell lung carcinoma, cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer,tumors of the biliary tract, Ewing's tumor, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, myelodysplastic disease, heavy chain disease,neuroendocrine tumors, Schwanoma, and other carcinomas, as well as headand neck cancer.

In one embodiment, the antibodies and fragments thereof provided hereinare useful in the treatment of diseases caused by infectious agents.Infectious agents include, but are not limited to, bacterial,mycological, parasitic, and viral agents. Examples of such infectiousagents include the following: staphylococcus, methicillin-resistantStaphylococcus aureus, Escherichia coli, streptococcaceae,neisseriaaceae, cocci, enterobacteriaceae, enterococcus,vancomycin-resistant enterococcus, cryptococcus, histoplasmosis,aspergillus, pseudomonadaceae, vibrionaceae, campylobacter,pasteurellaceae, bordetella, francisella, brucella, legionellaceae,bacteroidaceae, gram-negativebacilli, clostridium, corynebacterium,propionibacterium, gram-positive bacilli, anthrax, actinomyces,nocardia, mycobacterium, treponema, borrelia, leptospira, mycoplasma,ureaplasma, rickettsia, chlamydiae, candida, systemic mycoses,opportunistic mycoses, protozoa, nematodes, trematodes, cestodes,adenoviruses, herpesviruses (including, for example, herpes simplexvirus and Epstein Barr virus, and herpes zoster virus), poxviruses,papovaviruses, hepatitis viruses, (including, for example, hepatitis Bvirus and hepatitis C virus), papilloma viruses, orthomyxoviruses(including, for example, influenza A, influenza B, and influenza C),paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses,flaviviruses, bunyaviridae, rhabdoviruses, rotavirus, respiratorysyncitial virus, human immunodeficiency virus and retroviruses.Exemplary infectious diseases include but are not limited tocandidiasis, candidemia, aspergillosis, streptococcal pneumonia,streptococcal skin and oropharyngeal conditions, gram negative sepsis,tuberculosis, mononucleosis, influenza, respiratory illness caused byRespiratory Syncytial Virus, malaria, schistosomiasis, andtrypanosomiasis.

In one embodiment, the antibodies and fragments thereof provided hereinare useful in the treatment of diseases mediated by T-helper type 2(Th2) T cells, such as, for example, asthma, allergy, or graft versushost disease.

In one embodiment, the antibodies and fragments thereof provided hereinare useful in for the stimulation of an immune response in a subject inneed thereof. For example, in one embodiment, the anti-PD-L1 antibodiesand fragments thereof may be administered in conjunction with an antigenof interest for the purpose of eliciting an immune response to saidantigen. An antigen of interest may be an antigen associated with apathogen such as a virus or bacterium. Thus, in one embodiment, thepresent invention provides a vaccine comprising an anti-PD-L1 antibodyand an antigen, wherein the vaccine elicits an antigen-specific immuneresponse.

In one embodiment, the anti-PD-L1 antibodies provided herein modulateregulatory T cell function. CD4+ CD25+ regulatory T cells arelymphocytes that suppress or reduce the effects of effector T cellfunctions. The terms “regulatory T cell” and “Treg” are usedinterchangeably herein. In one embodiment, the anti-PD-L1 antibodiesprovided herein prevent or reverse the inhibitory effects of regulatoryT cells on effector T cell cytokine production. For example, in oneembodiment, the anti-PD-L1 antibodies provided herein restore thecapacity for IFNγ production to effector T cells in contact withregulatory T cells.

In one embodiment, the antibodies and fragments thereof disclosed hereinmay be administered to the subject by at least one route selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intratympanic,intrauterine, intravesical, intravitreal, bolus, subconjunctival,vaginal, rectal, buccal, sublingual, intranasal, intratumoral, andtransdermal.

In one embodiment, the antibodies and fragments thereof disclosed hereinmay be administered to a subject in need thereof in combination with oneor more additional therapeutic agent. In one embodiment, the antibodiesand fragments thereof may be administered to a subject before, during,and/or after administration to the subject of the additional therapeuticagent. In one embodiment, the additional therapeutic agent is achemotherapeutic agent, radiotherapeutic agent, cytokine, antibody orfragment thereof, or any other additional therapeutic that is indicatedfor the disease to be treated. In one embodiment, the anti-PD-L1antibody and the additional therapeutic agent exhibit therapeuticsynergy when administered together, whether concurrently orsequentially. In one embodiment, the anti-PD-L1 antibody and theadditional therapeutic agent are administered in separate formulations.In another embodiment, the anti-PD-L1 antibody and the additionaltherapeutic agent are administered in the same formulation. In oneembodiment, the anti-PD-L1 antibodies and fragments provided hereinenhance the immune modulating effect of the one or more additionaltherapeutic agent. In another embodiment, the one or more additionaltherapeutic agent enhances the effect of the anti-PD-L1 antibody orfragment thereof.

The present invention provides isolated antibodies and antigen bindingfragments thereof, and nucleic acids encoding such antibodies andfragments, as well as compositions comprising such isolated antibodies,fragments, and nucleic acids. The term “isolated” refers to a compoundof interest (e.g., an antibody or nucleic acid) that has been separatedfrom its natural environment. The present invention further providespharmaceutical compositions comprising the isolated antibodies orfragments thereof, or nucleic acids encoding such antibodies orfragments, and further comprising one or more pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers include, forexample, excipients, diluents, encapsulating materials, fillers,buffers, or other agents.

The use of the singular includes the plural unless specifically statedotherwise. The word “a” or “an” means “at least one” unless specificallystated otherwise. The use of “or” means “and/or” unless statedotherwise. The meaning of the phrase “at least one” is equivalent to themeaning of the phrase “one or more.” Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. Also, terms such as “element” or “component” encompassboth elements or components comprising one unit and elements orcomponents comprising more than one unit unless specifically statedotherwise.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of non-critical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1: Generation of hPD-L1 Monoclonal Antibody

Immunization of Mice with hPD-L1-HisTag and hPD-L1-mFc

To generate antibodies against the human PD-L1, cDNAs encoding the openreading frame of the extracellular domain of hPD-L1 fused with ahistidine tag (hPD-L1-HisTag, SEQ ID NOs:143 and 144), mouse Fc(hPD-L1-mFc, SEQ ID NOs:145 and 146), and human Fc tag (hPD-L1-hFc, SEQID NO:147 and 148) were obtained by PCR and subcloned into expressionvector pcDNA3.1 (Invitrogen CAT #:V-790), respectively. After transientexpression in freestyle 293 cells, hPD-L1-HisTag was purified with NTAcolumn (GE healthcare), hPD-L1-mFc and hPD-L1-hFc were purified withProtein G column (GE healthcare).

BALB/cJ mice were immunized subcutaneously every 2 weeks for 6 weekswith recombinant hPD-L1-HisTag protein (100 μg/mouse) or hPD-L1-mFcemulsified with an equal volume of Freund's complete/incompleteadjuvant. Three days before fusion, mice were boosted by intravenousinjection of the antigen without adjuvant. Spleen cells (1×10⁸) fromimmunized mouse were fused with SP2/0 myeloma cells (1.5×10⁷) with PEGHybri-Max (Sigma Inc., CAT #:7181). After fusion, the cells weredistributed into 96-well plates at 0.1 ml per well and incubated at 37°C., 5% CO₂ incubator. On day 1, cells were fed by adding an additional0.1 ml per well with media containing serum and HAT plus 2×methotrexate.On day 3 and day 7, 0.1 ml of media from each well was replaced with 0.1ml of fresh HT media. The screening typically occurred between days9-14, and culture supernatant was tested for antibody reacting withhPD-L1-hFc by ELISA.

To clone the selected hybridoma cell, limiting dilution was carried outfour times. The hybridoma cells were cultured in Dulbecco's ModifiedEagle's medium (GIBCO; Invitrogen Corporation, Carlsbad, Calif.)containing 10% fetal calf serum, 1% penicillin/streptomycin, 2%L-glutamine, and 1% adjusted NaHCO₃ solution. The selected hybridomacells were then adapted in serum free culture medium and the antibodywas purified from the supernatant using Protein-G column (GEhealthcare). After washing with PBS, bound antibodies were eluted using0.1 M Glycine pH3.0, followed by pH neutralization using 2.0 M Tris.Ultra-15 centrifugal concentrators (Amicon) were used for bufferexchanging and antibody concentrating.

Example 2: Anti-PD-L1 Antibodies cDNA Sequences Cloning and HumanizationCloning of Immunoglobulin cDNAs

Total RNA isolated from the hybridoma cell line producing hPD-L1antibody by RNeasy Mini Kit (Qiagen, CAT #:74104) was used as thetemplate to synthesize first-strand cDNA with SuperScript® II ReverseTranscriptase (Life Technology, CAT #18064-14) according to themanufacturer's instructions. The cDNA product was then subjected to PCRin a 50 μl volume reaction mixture using degenerate mouse IgG primers(Kettleborough Calif., et al, European Journal of Immunology 23: 206-211(1993), Strebe N, et al, Antibody Engineering 1:3-14 (2010)). Thereaction was carried out in a S1000™ Thermal Cycler (Bio-Rad, CAT#:184-2000) with 30 cycles of: 94° C., 1.5 minutes for denaturation; 50°C., 1 minutes for annealing; and 72° C., 1 minute for synthesis. At theend of the 30th cycle, the reaction mixture was incubated another 7minutes at 72° C. for extension.

The PCR mixture was subjected to electrophoresis in a 1%agarose/Tris-Borate gel containing 0.5 μg/ml ethidium bromide. DNAfragments having the expected sizes (approximately 450 bp for the heavychain and the light chain) were excised from the gel and purified. 3 μlof purified PCR product were cloned into the pMD-18T vector (Takara, CAT#:D101A) and transformed into One Shot® TOP10 chemically competent E.coli (Invitrogen, CAT #:C4040-03). Clones were screened by colony PCRusing universal M13 forward and reverse primers, and 10 positive clonesfrom each reaction were chosen for DNA sequencing in both directionsusing M13 forward and M13 reverse primers.

The heavy and light variable region sequences of antibodies m4A8 (SEQ IDNOs: 25-28), m4D1 (SEQ ID NOs: 21-24), m5G9 (SEQ ID NOs: 5-8), m5G11(SEQ ID NOs: 9-12), m8C6 (SEQ ID NOs: 13-16), m8H3 (SEQ ID NOs: 33-36),m8H4 (SEQ ID NOs: 29-32), m7B4 (SEQ ID NOs: 17-20), m13C5 (SEQ ID NOs:1-4) and m15F1 (SEQ ID NOs: 37-40) were amplified from the correspondinghybridoma clones. These antibodies showed desired functions, such asblocking PD-L1 binding to PD-1, and enhanced T cell activation andcytokine release.

Construction and Expression of Chimeric 5G11 and 13C5 Antibody

8C6, 8H4, 5G11 and 13C5 chimeric light chains (SEQ ID NOs: 52, 56, 62,and 68, respectively) were constructed by linking the PCR-cloned cDNAsof mouse VL regions to human kappa chain constant region, respectively.8C6, 8H4, 5G11 and 13C5 chimeric heavy chains (SEQ ID NOs: 50(8C6-IgG4), 54 (8H4-IgG4), 58 (5G11-IgG1), 60 (5G11-IgG4), 64(13C5-IgG1), and 66 (13C5-IgG4)) were constructed by linking thePCR-cloned cDNAs of mouse VH regions to human IgG1 and IgG4 constantregions. The 5′ends of the mouse cDNA sequences were modified using PCRprimers designed to add a leader sequence to both light chain and heavychain.

Freestyle 293 cells (200 mL at 10⁶/mL) were transfected with 100 μg ofeach of the chimeric heavy and light chain expression plasmids andcultured for 6 days. The chimeric antibody in the supernatant was thenpurified with Protein-G column (GE healthcare). Binding of the chimericantibody with PD-L1 was measured by ELISA and Biacore, and was shown tobind to PD-L1 with comparable affinity to that of the murine parentantibody.

Antibody Humanization Design

5G11 and 13C5 antibodies were humanized using CDR grafting approach(see, for example, U.S. Pat. No. 5,225,539). The light chain and heavychain variable chain sequences of the murine antibody 5G11 and 13C5 werecompared to those available in the Research Collaboratory for StructuralBioinformatics (RCSB) protein databank(http://www.ncbi.nlm.nih.gov/igblast/igblast.cgi). The model of 5G11 and13C5 were generated respectively based on the VH and VL structure withthe highest sequence homology.

The template human antibodies to be grafted with the complementarydetermining regions (CDRs) in the VH and VL of mouse 5G11 and 13C5antibody were selected from human antibody germlines having highsequence homology with mouse 5G11 and 13C5 antibody by searching theinternational immunogenetics information system website(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi). For 5G11,the template human VH selected was a combination of IGHV2-5*10 andIGHJ4*01, and template human VL selected was a combination ofIGKV1-33*01 and IGKJ2*01. For 13C5, the template human VH selected was acombination of IGHV3-21*04 and IGHJ4*01, and template human VL selectedwas a combination of IGKV7-3*01 and IGKJ2*01.

CDR amino acid sequences of the aforementioned template human antibodieswere substituted by the CDRs of hybridoma (mouse) 5G11 (SEQ ID NOs93-98) and 13C5 (SEQ ID NOs 81-86) antibodies. The frameworks of theabove-mentioned template human antibody VH and VL were grafted with thenecessary amino acid sequences from VH and VL of mouse 5G11 and 13C5antibody to give a functional humanized antibody. As for VH and VL of5G11 and 13C5, several sites of framework amino acid of theaforementioned template human antibody were backmutated to thecorresponding amino acid sequences in mouse 5G11 and 13C5 antibody. Forthe light chain variable region of humanized 5G11 antibody, the aminoacid at position 60 is mutated from Ser (S) to Asp (D), and the aminoacid at position 67 is mutated from Ser (S) to Tyr (Y); and for theheavy chain variable region of humanized 5G11 antibody, the amino acidat position 24 is mutated from Phe (F) to Val (V), the amino acid atposition 49 is mutated from Ala (A) to Gly (G), the amino acid atposition 73 is mutated from Thr (T) to Asn (N), and the amino acid atposition 83 is mutated from Thr (T) to Asn (N). For the light chainvariable region of humanized 13C5, the amino acid at position 53 ismutated from Tyr (Y) to Lys (K); and for the heavy chain variable regionof humanized 13C5, the amino acid at position 28 is mutated from Thr (T)to Ile (I), the amino acid at position 30 is mutated from Ser (S) to Arg(R), the amino acid at position 49 is mutated from Ser (S) to Ala (A),and the amino acid at position 94 is mutated from Tyr (Y) to Asp (D).The amino acid sequences of VH and VL of humanized 5G11 are provided asSEQ ID NOs:42 and 44, respectively; DNA sequences encoding the VH and VLof humanized 5G11 are provided as SEQ ID NOs: 41 and 43, respectively.The amino acid sequences of VH and VL of humanized 13C5 are provided asSEQ ID NOs: 46 and 48, respectively); DNA sequences encoding the VH andVL of humanized 13C5 are provided as SEQ ID NOs: 45 and 47,respectively.

The amino acid sequences of the full light chain for humanizedantibodies 5G11 and 13C5 are provided as SEQ ID NOs: 74 and 80,respectively. The DNA sequences encoding the full length humanized 5G11and 13C5 are provided as SEQ ID NOs: 73 and 79, respectively. IgG1 andIgG4 versions of the humanized 5G11 and 13C5 antibodies were produced.The IgG1 constant region carries D265A mutation (Clynes R, et al, NatureMedicine 6: 443-446 (2000)), while IgG4 constant region has F234A andL235A double mutation (Xu D, et al, Cellular Immunology 200: 16-26(2000)). The DNA and amino acid sequences for the full length IgG1 heavychain of humanized antibody 5G11-hIgG1 are provided as SEQ ID NOs: 69and 70, respectively. The DNA and amino acid sequences for the fulllength IgG4 heavy chain of humanized antibody 5G11-hIgG4 are provided asSEQ ID NOs: 71 and 72, respectively. The DNA and amino acid sequencesfor the full length IgG1 heavy chain of humanized antibody 13C5-hIgG1are provided as SEQ ID NOs: 75 and 76, respectively. The DNA and aminoacid sequences for the full length IgG4 heavy chain of humanizedantibody 13C5-hIgG4 are provided as SEQ ID NOs: 77 and 78, respectively.

Construction and Expression of Humanized 5G11 and 13C5 Antibody

DNA encoding humanized 5G11 and 13C5 antibody light chain and heavychain was synthesized and cloned to the expression vector pcDNA3.1(Invitrogen, CAT: #V-790). Freestyle 293 cells (200 mL at 10⁶/mL) weretransfected with 100 μg of each of the humanized heavy and light chainexpression plasmids and cultured for 6 days. The humanized antibody inthe supernatant was then purified with Protein-G column (GE healthcare).

The binding kinetics between PD-L1 and PD-L1 antibodies were measured byBiacore analysis, which was performed at 25° C. on a Biacore3000instrument and recorded with a data collection rate of 1 Hz. Polyclonalrabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10 mM pH 5.0sodium acetate and immobilized onto reference and experiment flow cellsof a CM5 biosensor chip to around 15000RU using an amine coupling kit(GE, BR10050). In the beginning of each cycle, diluted test antibody(1.5 μg/mL) was injected over experiment flow cell for 1 minute to becaptured. PD-L1 analyte series were prepared by diluting the stocks withrunning buffer to 100 nM followed by 2× serial dilution in the samebuffer down to 0.78 nM. Analytes were injected in series over thereference and experiment flow cells for 3 minutes at a flow rate of 30μL/minute. Running buffer (PBS with 0.05% P20) was allowed to flow overfor 10 minutes at a flow rate of 30 μL/minute. At the end of each cycle,the biosensor surface was regenerated with 3 minutes injection of 10 mMpH1.7 Glycine-HCl buffer at a flow rate of 10 μL/minute. For eachanalyte sample injection (i.e. each cycle), binding responses obtainedfrom the experimental biosensor surface were double referenced bysubtracting simultaneously recorded responses from the reference surfacefollowed by additional subtraction of responses from a single referencedrunning buffer sample. The association and dissociation rate constants(ka and kd) were determined simultaneously by fitting double-referencedsensorgrams of the entire titration series to Langmuir model (1:1) usingBiaevaluation 4.0 software. The dissociation constant, KD, wascalculated from the determined rate constants by the relation KD=kd/ka.The binding affinity of anti-PD-L1 antibodies with human PD-L1 andcynomolgus PD-L1 (cyno-PD-L1) are summarized in Table 4.

TABLE 4 PD-L1 binding affinity of anti-PD-L1 antibodies SelectedAntibody Antigen KD (M) m4A8 Human PD-L1 2.33E−9 m4D1 Human PD-L14.39E−9 m5G9 Human PD-L1 4.78E−9 m5G11 Human PD-L1 1.90E−10 m7B4 HumanPD-L1 6.01E−9 m8H3 Human PD-L1 6.60E−9 m8H4 Human PD-L1 4.56E−9 m8C6Human PD-L1 1.53E−9 m13C5 Human PD-L1 1.35E−9 m15F1 Human PD-L1 3.59E−9ch5G11 Human PD-L1 2.86E−10 ch13C5 Human PD-L1 2.28E−09 hu5G11 HumanPD-L1 2.25E−10 hu13C5 Human PD-L1 1.74E−09 hu5G11 Cyno-PD-L1 2.75E−10hu13C5 Cyno-PD-L1 2.43E−09

Example 3: ELISA Based Binding Analysis of Anti-PD-L1 Antibodies

ELISA binding analyses were conducted based on human PD-L1-mFc (forchimeric and humanized antibody detection) and PD-L1-hFc protein (forhybridoma antibody detection). 96-well plates (Costar, Cat No: 9018)were coated with 100 μL of 2 μg/ml PD-L1-mFc (Crownbio) in coatingbuffer PBS (Hyclone, Cat No:SH30256.01B) overnight at 4° C. The wellswere aspirated and non-specific binding sites were blocked by adding 200μL of blocking buffer (PBS with 1% (w/v) of bovine serum albumin (BSA,Roche, Cat No:738328)) and incubating for 1 hour at 37° C. After theplates were washed three times with wash buffer (PBS with 0.05% (v/v)Tween20 (Sigma, Cat No:P1379)), 100 μL/well of 1:10 serial dilutions ofhybridoma (FIG. 1), chimeric (FIG. 2), or humanized (FIG. 3) anti-PD-L1antibodies in blocking buffer (starting from 20 μg/mL) were added andincubated at room temperature for 1 hour. The plates were washed andincubated with 100 μL/well of Goat anti-Mouse IgG (H+L) (Thermo, Cat No:31432) in blocking buffer for 60 min. After the plates were washed, 100μL/well of substrate solution TMB (eBioscience, Cat No: 00-4201-56) wereadded and the plates were incubated for 2 min at room temperature. 100μL/well of stop solution (2N H2504) was added to stop the reaction. Thecolorimetric signals were developed and read at 450 nm using an AutoPlate SpectraMax Plus (Supplier: Moleculer Devices; Model: MNR0643;Software: SoftMax Pro v5.4). Data were analyzed using GraphPad Prism 5and EC50 was calculated (FIGS. 1-3; Tables 5-7). These data demonstratedthat anti-PD-L1 antibodies (hybridoma, chimeric, and humanized) bindPD-L1, as measured by ELISA.

TABLE 5 ELISA based binding EC50 of anti-PD-L1 hybridoma monoclonalantibody with PD-L1 hybridoma Ab m5G11 m7B4 m4D1 m8H4 m13C5 EC50 ng/ml45.9 31.42 7.14 29.04 65.1 hybridoma Ab m8C6 m5G9 m4A8 m8H3 m15F1 EC50ng/ml 18.2 31.2 57.6 48.7 48.7

TABLE 6 ELISA based binding EC50 of anti-PD-L1 chimeric antibody withPD-L1 ch5G11 ch5G11 ch8C6 ch8H4- ch13C5 ch13C5 Chimeric Ab hIgG1 hIgG4hIgG4 hIgG4 hIgG1 hIgG4 EC50 ng/ml 82.1 90 76 133.6 72.1 118

TABLE 7 ELISA based binding EC50 of humanized anti-PD-L1 antibody withPD-L1 Humanized Ab hu13C5- hu13C5- hu5G11- hu5G11- hIgG1 hIgG4 hIgG1hIgG4 EC50 85.6 126.82 49.5 69.9 (ng/ml)

ELISA based ligand blockage analyses were conducted via blockingbiotinylated human PD-L1-mFc's binding to human PD-1-hFc. PD-1-hFcantigen (Crownbio) was suspended in PBS buffer (2 ug/ml, 100 ul/well)and coated on the 96 well plate (Costar, Cat No: 9018) 4° C. overnight.The wells were aspirated and non-specific binding sites were blocked byadding 200 μL of blocking buffer (PBS with 1% (w/v) of bovine serumalbumin (BSA, Roche, Cat No:738328)) and incubating for 1 hour at 37° C.After the plate was washed three times with wash buffer (PBS with 0.05%(v/v) Tween20 (Sigma, Cat No:P1379)), 100 μL/well of 1:3 serialdilutions of hybridoma (FIG. 4), chimeric (FIG. 5), or humanized (FIG.6) anti-PD-L1 antibodies in blocking buffer (starting from 20 μg/mL)were added and incubated at 37° C. for 1 hour. 100 μl PDL-1-mFc-biotin(0.1 μg/ml) was then added to each well and incubated at 37° C. for 2 h.After the plate was washed 3 times, secondary antibody (Avidin HRPeBioscience cat No.:E07418-1632, 1:500, 100 μl/well) was added andincubated at 37° C. for 0.5 hour. After the plate was washed, 100μL/well of substrate solution TMB (eBioscience, Cat No: 00-4201-56) wasadded and the plate was incubated for 3 min at room temperature. 100μL/well of stop solution (2N H₂SO₄) was added to stop the reaction. Thecolorimetric signals were developed and read at 450 nm using an AutoPlate SpectraMax Plus (Supplier: Moleculer Devices; Model: MNR0643;Software: SoftMax Pro v5.4). Data were analyzed using GraphPad Prism 5and IC50 was calculated (FIGS. 4-6; Tables 8-10). These datademonstrated that anti-PD-L1 antibodies (hybridoma, chimeric, andhumanized) can block PD-1's binding with PD-L1 on the cell surface, asmeasured by ELISA.

TABLE 8 IC50 of anti-PD-L1 hybridoma monoclonal antibody inhibiting PD-1binding with PD-L1 on solid surface Hybridoma Ab m5G11 m7B4 m4D1 m8H4m13C5 m8C6 m5G9 m4A8 m8H3 m15F1 IC50 710.2 892.0 332.2 787.8 871.7 343.7613.2 867.8 647.4 655.3 (ng/ml)

TABLE 9 IC50 of anti-PD-L1 chimeric antibody inhibiting PD-1 bindingwith PD-L1 on solid surface ch5G11- ch5G11- ch8C6- ch8H4- ch13C5-ch13C5- Chimeric Ab hIgG1 hIgG4 hIgG4 hIgG4 hIgG1 hIgG4 IC50 (ng/mL)1006 926.1 476.6 848.1 805.2 375.3

TABLE 10 IC50 of humanized anti-PD-L1 antibody inhibiting PD-1 bindingwith PD-L1 on solid surface Humanized Ab hu5G11- hIgG1 hu5G11-hIgG4hu13C5-hIgG1 hu13C5-hIgG4 IC50 793.6 822.5 1202.6 1192.4 (ng/ml)

Example 4: Cell-Based Binding Analysis of Anti-PD-L1 Antibodies

Cell binding analyses of anti-PD-L1 antibodies were performed based onbinding to a 293T cell line stably expressing PD-L1 (PD-L1-293T). 2×10⁵293 T-PD-L1 cells were added into each well of 96-well culture platesand incubated with the indicated antibody (20 μg/ml with the dilution of1:5) at 4° C. for 1 h. After the cells were washed three times with FACSbuffer, the secondary antibody (PE Goat anti-mouse: 1:200; PE mouseanti-human: 1:10) was added to the cells at 100 μl/well, and incubatedat 4° C. for 40 min. Cells were washed three times with FACS buffer andanalyzed by FACS Array. Binding of hybridoma antibodies is shown inFIGS. 7a and 7b . Binding of chimeric antibodies is shown in FIG. 8.Binding of humanized antibodies is shown in FIG. 9. The calculated EC50for hybridoma, chimeric, and humanized antibodies are shown below inTables 11, 12, and 13, respectively. These data demonstrated thatanti-PD-L1 antibodies (hybridoma, chimeric, and humanized) bind PD-L1,as measured by FACS analysis.

TABLE 11 EC50 of anti-PD-L1 hybridoma monoclonal antibody with the PD-L1on cell surface Hybridoma Ab m4D1 m4A8 m5G11 m8H4 m8H3 EC50 ng/ml 36.0767.83 35.94 43.49 50.81 Hybridoma Ab m8C6 m9G9 m7B4 m13C5 m15F1 EC50ng/ml 40.97 33.7 47.41 45.29 47.8

TABLE 12 EC50 of anti-PD-L1 chimeric antibody with the PD-L1 on cellsurface Chimeric Ab ch13C5 hIgG1 ch5G11 hIgG1 ch5G11 hIgG4 EC50 ng/ml75.75 58.26 89.68

TABLE 13 EC50 of humanized anti-PD-L1 antibody with the PD-L1 on cellsurface Humanized Ab hu5G11- hu5G11- hIgG1 hIgG4 hu13C5-hIgG1hu13C5-hIgG4 EC50 ng/ml 47.93 54.33 80.01 80.39

The effect of anti-PD-L1 antibody on PD-1 binding to PD-L1 on the cellsurface was also investigated. Briefly, PD-L1-293T cells were suspendedin FACS buffer (PBS with 3% fetal calf serum). Various concentrations ofthe hybridoma (FIG. 10), chimeric (FIG. 11), or humanized (FIG. 12)anti-PD-L1 antibodies were added to the cell suspension and incubated at4° C. for 60 minutes in 96 well plates. Biotin-labeled PD-L1 protein wasthen added to the wells and incubated at 4° C. for 60 minutes. The cellswere washed 3 times with PBS and incubated with mouse anti-biotin PE(Biolegend, cat #409004). The cell-associated fluorescence was thendetected by flow cytometry analysis using FACS array. The effects ofanti-PD-L1 antibodies on PD-1 binding with PD-L1-293T were measured bythe mean fluorescent intensity (MFI) of staining. Inhibition of PD-1binding by anti-PD-L1 hybridoma antibodies is shown in FIGS. 10a and 10b. Inhibition of PD-1 binding by anti-PD-L1 chimeric antibodies is shownin FIG. 11. Inhibition of PD-1 binding by anti-PD-L1 humanizedantibodies is shown in FIG. 12. The calculated IC50 for the hybridoma(Table 14), chimeric (Table 15), and humanized (Table 16) antibodies areshown in the tables below. These data demonstrated that anti-PD-L1antibodies (hybridoma, chimeric, and humanized) can block PD-1's bindingwith PD-L1 on the cell surface, as measured by FACS analysis.

TABLE 14 IC50 of anti-PD-L1 hybridoma monoclonal antibody inhibitingPD-1 binding with PD-L1 on cell surface Hybridoma Ab mIgG1 m4D1 m5G11m13C5 m7B4 m8H4 IC50 ng/ml NA 27.3 16.3 28.9 38.1 30.6 Hybridoma Ab m4A8m5G9 m8C6 m8H3 m15F1 IC50 ng/ml 29.1 49.1 8.2 33.6 21.1

TABLE 15 IC50 of anti-PD-L1 chimeric antibody inhibiting PD-1 bindingwith PD-L1 on cell surface Chimeric ch5G11- ch5G11- ch8C6- ch8H4-ch13C5- ch13C5- Ab hIgG1 hIgG4 hIgG4 hIgG4 hIgG1 hIgG4 IC50 ng/ml 40.3633.18 34.91 42.02 42.71 35.78

TABLE 16 IC50 of humanized anti-PD-L1 antibody inhibiting PD-1 bindingwith PD-L1 on cell surface Humanized Ab hu13C5- hu13C5- hu5G11- hu5G11-hIgG4 hIgG1 hIgG4 hIgG1 hIgG4 IC50 ng/ml NA 18.5 49.9 16.5 9.6

Example 5: Effect of Anti-PD-L1 Antibodies on T Cell Activation in aMixed Lymphocyte Reaction

A mixed lymphocyte reaction was employed to demonstrate the effect ofmurine (FIG. 13a, 13b ), chimeric (FIG. 14a, 14b ), or humanized (FIG.15a, 15b ) anti-PD-L1 antibodies in blocking the PD-L1/PD-1 pathway inlymphocyte effector cells. T cells in the assay were tested for IFN-γand IL-2 secretion in the presence or absence of humanized anti-PD-L1antibody.

Human CD4⁺ T-cells were purified from human PBMC using a CD4⁺ negativeselection isolation kit (Mitenyi Biotech, cat #130-091-155). Immaturedendritic cells (DC) were derived from monocytes isolated from humanPBMC using the Mo-DC Generation Toolbox (Miltenyi, Cat #130-093-568).The cells were cultured with Mo-DC Differentiation Medium for 7 days,and were then induced to be mature DC with Mo-Dc Maturation medium for 2days. To set up the MLR, for each reaction, 10⁵ purified T-cells and 10⁴allogeneic mature DC cells were added in a total volume of 200 pl. Thetesting antibody was assayed at different concentrations as shown inFIGS. 13a, 13b, 14a, 14b, 15a, and 15b (i.e., 20 μg/mL, 2 μg/mL, 0.2μg/mL, 0.02 μg/mL, and 0.002 μg/mL). Either no antibody or an isotypecontrol antibody was used as a negative control. The cells were culturedfor 5 days at 37° C. On day 6^(th), the levels of IFN-γ and IL-2 in theculture medium were measured using the IL-2 ELISA kit (eBioscience) andhIFN-γ ELISA kit (R&D, cat #DY285). The results are shown in FIGS. 13a,14a, and 15a for IL-2 secretion, and FIGS. 13b, 14b, and 15b for IFN-γsecretion. The results of the study showed that hybridoma, chimeric, andhumanized anti-PD-L1 antibodies promoted T-cell IFN-γ and IL-2 secretionin a concentration dependent manner. In contrast, cultures containingthe isotype control antibody did not show increase in IFN-γ and IL-2secretion.

Example 6: Effect of Anti-PD-L1 Antibody on the Function of T RegulatoryCells

T regulatory cells (CD4+, CD25+) are lymphocytes that suppress theimmune response. The effect of T regulatory cells on cytokine secretionof T effector cells in MLR was tested in the presence or absence ofchimeric or humanized anti-PD-L1 antibodies. T regulatory cells (CD4+CD25+) were purified from PBMC using a regulatory T cell isolation kit(Miltenyi Biotec, cat #130-091-301). Immature dendritic cells (DC) werederived from monocytes isolated from human PBMC using the Mo-DCGeneration Toolbox (Miltenyi, cat #130-093-568). The cells were culturedwith Mo-DC Differentiation Medium for 7 days, and were then induced tobe mature DC with Mo-Dc Maturation medium for 2 days. T regulatory cellswere added into a mixed lymphocyte reaction containing purified CD4⁺CD25⁻ T cells and allogeneic dendritic cells in a 4:1 ratio of CD4⁺CD25⁻ to T regulatory cells. For example: the reaction was added with1×10{circumflex over ( )}5 cells/well of CD4⁺CD25⁻ cells,1×10{circumflex over ( )}4 cells/well of mDC, and 0.25×10{circumflexover ( )}5 cells/well of CD4⁺ CD25⁺ cells. Antibody was added to eachreaction at a concentration of 10 μg/ml. Either no antibody or anisotype control antibody was used as a negative control. The cells werecultured for 5 days at 37° C. On the 5^(th) day, 50 μl medium was takento detect IL-2 and IFN-gamma concentration. After supplementing eachwell with 50 μl culture medium, the cells were cultured for another 2days before analyzed for cell proliferation by CTG (Promega, G7573). Thelevels of IFN-γ and IL-2 in the culture medium were measured using ahIFN-γ ELISA kit (R&D, cat #DY285) and IL-2 ELISA kit (eBioscience). Asshown in FIG. 16, chimeric and humanized anti-PD-L1 antibodies,ch-13C5-hIgG1, ch-13C5-hIgG4, hu-13C5-IgG1, hu-13C5-IgG4, ch-5G11-IgG1,ch-5G11-IgG4, hu-5G11-IgG1, and hu-5G11-IgG4, can reduce the inhibitoryeffect of Treg cells on the secretion of IFN-γ by CD4⁺CD25⁻ T effectorcells, suggesting that anti-PD-L1 antibodies can modulate the immunesuppression function of T regulatory cells.

Example 7: Effect of Humanized Anti-PD-L1 Antibody on Autologous T CellActivation

In this example, the effect of blocking PD-1/PD-L1 pathway by anti-PD-L1antibody on T cell activation was examined. Purified human CD4+ T cells(Mitenyi Biotech, cat #130-091-155) were activated with 1 μg/ml solubleanti-CD3 antibody (R&D, cat #MAB100) in the presence of autologousmonocyte-derived dendritic cells (DCs). After three days of activationin the presence or absence of titrated anti-PD-L1 antibody, culturemedium was harvested and the concentration of IFNγ was measured withELISA. The results are shown in FIG. 17 and suggest that PD-L1 blockageby humanized anti-PD-L1 antibodies enhanced IFN-γ secretion by T cells.

Example 8: Human Recall T Cell Response to Tetanus Toxoid Challenge isEnhanced by Humanized Anti-PD-L1 Antibody

To investigate whether the antigen-specific T cell receptor triggeringwas modulated by blocking PD-1/PD-L1 pathway with anti-PD-L1 antibodies,the human T-cell recall assay was employed using tetanus toxoid (TT)antigen to stimulate pre-existing memory T cells in the blood of healthyTT immunized donors. To this end, fresh PBMC from recently [<1 year] TTimmunized donors were plated into 96-well round bottom plates (costar,cat #3799) at 4×10{circumflex over ( )}5 cells/well using RPMI1640(Invitrogen, cat # A10491-01) supplemented with 80 U/ml penicillin, 80g/ml streptomycin and 30% autologous serum, added with humanized 5G11 or13C5 at various concentrations, and stimulated with 0.1 ug/ml SEB and 1μg/ml TT (Astarte Biologies). After co-culture for 7 days at 37° C., 5%CO₂, the supernatant was harvested and the concentration of IFN-γ wasmeasured. FIGS. 18a and 18b provide the results of the assay using PBMCfrom two separate donors. The results of the study demonstrate that,compared to TT antigen alone, PD-L1 blockage with anti-PD-L1 antibodyresulted in enhanced IFN-γ secretion by memory T cells.

In summary, the humanized 5G11 and 13C5 antibody retained the functionalactivity of their parental antibodies during the humanization process.

1. A method for treating cancer in a subject in need thereof, the methodcomprising administering to the subject an antibody or fragment thereofthat binds to PD-L1, wherein the antibody or fragment thereof comprises:(i) a heavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:93, 94, and 95, respectively, and a light chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 96, 97, and 98, respectively; (ii) aheavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 81, 82,and 83, respectively, and a light chain CDR1, CDR2, and CDR3 sequencecomprising SEQ ID NO: 84, 85, and 86, respectively; (iii) a heavy chainCDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 87, 88, and 89,respectively, and a light chain CDR1, CDR2, and CDR3 sequence comprisingSEQ ID NO: 90, 91, and 92, respectively; (iv) a heavy chain CDR1, CDR2,and CDR3 sequence comprising SEQ ID NO: 99, 100, and 101, respectively,and a light chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:102, 103, and 104, respectively; (v) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 105, 106, and 107, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 108,109, and 110, respectively; (vi) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 111, 112, and 113, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 114,115, and 116, respectively; (vii) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 117, 118, and 119, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 120,121, and 122, respectively; (viii) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 123, 124, and 125, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 126,127, and 128, respectively; (ix) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 129, 130, and 131, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 132,133, and 134, respectively; or (x) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 135, 136, and 137, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 138,139, and 140, respectively.
 2. The method of claim 1, wherein theantibody or fragment thereof comprises a heavy chain CDR1, CDR2, andCDR3 comprising an amino acid sequence according to SEQ ID NOs: 93, 94,and 95, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence according to SEQ ID NOs: 96, 97, and 98,respectively.
 3. The method of claim 1, wherein the antibody or fragmentthereof comprises a heavy chain CDR1, CDR2, and CDR3 comprising an aminoacid sequence according to SEQ ID NOs: 81, 82, and 83, respectively; anda light chain CDR1, CDR2, and CDR3 comprising an amino acid sequenceaccording to SEQ ID NOs: 84, 85, and 86, respectively.
 4. The method ofclaim 1, wherein the antibody or fragment thereof is humanized.
 5. Themethod of claim 1, wherein the antibody or fragment thereof comprises(i) a heavy chain variable region comprising SEQ ID NO: 2 and a lightchain variable region comprising SEQ ID NO: 4; (ii) a heavy chainvariable region comprising SEQ ID NO: 6 and a light chain variableregion comprising SEQ ID NO: 8; (iii) a heavy chain variable regioncomprising SEQ ID NO: 10 and a light chain variable region comprisingSEQ ID NO: 12; (iv) a heavy chain variable region comprising SEQ ID NO:14 and a light chain variable region comprising SEQ ID NO: 16; (v) aheavy chain variable region comprising SEQ ID NO: 18 and a light chainvariable region comprising SEQ ID NO: 20; (vi) a heavy chain variableregion comprising SEQ ID NO: 22 and a light chain variable regioncomprising SEQ ID NO: 24; (vii) a heavy chain variable region comprisingSEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO:28; (viii) a heavy chain variable region comprising SEQ ID NO: 30 and alight chain variable region comprising SEQ ID NO: 32; (ix) a heavy chainvariable region comprising SEQ ID NO: 34 and a light chain variableregion comprising SEQ ID NO: 36; (x) a heavy chain variable regioncomprising SEQ ID NO: 38 and a light chain variable region comprisingSEQ ID NO: 40; (xi) a heavy chain variable region comprising SEQ ID NO:42 and a light chain variable region comprising SEQ ID NO: 44; or (xii)a heavy chain variable region comprising SEQ ID NO: 46 and a light chainvariable region comprising SEQ ID NO:
 48. 6. The method of claim 1,wherein the antibody or fragment thereof comprises a heavy chainvariable region according to SEQ ID NO: 42 and a light chain variableregion according to SEQ ID NO:
 44. 7. The method of claim 1, wherein theantibody or fragment thereof comprises a heavy chain variable regionaccording to SEQ ID NO: 46 and a light chain variable region accordingto SEQ ID NO:
 48. 8. The method claim 1, wherein the antibody orfragment thereof comprises a heavy chain according to SEQ ID NO: 70 anda light chain according to SEQ ID NO:
 74. 9. The method of claim 1,wherein the antibody or fragment thereof comprises a heavy chainaccording to SEQ ID NO: 72 and a light chain according to SEQ ID NO: 74.10. The method of claim 1, wherein the antibody or fragment thereofcomprises a heavy chain according to SEQ ID NO: 76 and a light chainaccording to SEQ ID NO:
 80. 11. The method of claim 1, wherein theantibody or fragment thereof comprises a heavy chain according to SEQ IDNO: 78 and a light chain according to SEQ ID NO:
 80. 12. The method ofclaim 1, wherein the antibody or fragment thereof is selected from thegroup consisting of a monoclonal antibody, an scFv, a Fab fragment, anFab′ fragment, and an F(ab)′ fragment.
 13. The method of claim 1,wherein the antibody or fragment thereof is linked or conjugated to atherapeutic agent.
 14. The method of claim 13, wherein the therapeuticagent is a cytotoxic drug, a radioactive isotope, an immunomodulator, oran antibody.
 15. The method of claim 1, wherein the method furthercomprises administering the antibody or fragment thereof to the subjectin combination with an additional therapeutic agent.
 16. The method ofclaim 1, wherein the cancer is a PD-L1 expressing cancer.
 17. The methodof claim 1, wherein the cancer is selected from the group consisting oflymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, coloncancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer,liver cancer, stomach cancer, rectal cancer, testicular cancer, salivarycancer, thyroid cancer, thymic cancer, epithelial cancer, head or neckcancer, gastric cancer, and pancreatic cancer; or any combinationthereof.
 18. A method for reducing PD-L1 expressing tumors or inhibitingthe growth of PD-L1 expressing tumor cells in a subject, the methodcomprising administering to the subject an antibody or fragment thereofthat binds to PD-L1, wherein the antibody or fragment thereof comprises:(i) a heavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:93, 94, and 95, respectively, and a light chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 96, 97, and 98, respectively; (ii) aheavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 81, 82,and 83, respectively, and a light chain CDR1, CDR2, and CDR3 sequencecomprising SEQ ID NO: 84, 85, and 86, respectively; (iii) a heavy chainCDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 87, 88, and 89,respectively, and a light chain CDR1, CDR2, and CDR3 sequence comprisingSEQ ID NO: 90, 91, and 92, respectively; (iv) a heavy chain CDR1, CDR2,and CDR3 sequence comprising SEQ ID NO: 99, 100, and 101, respectively,and a light chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:102, 103, and 104, respectively; (v) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 105, 106, and 107, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 108,109, and 110, respectively; (vi) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 111, 112, and 113, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 114,115, and 116, respectively; (vii) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 117, 118, and 119, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 120,121, and 122, respectively; (viii) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 123, 124, and 125, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 126,127, and 128, respectively; (ix) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 129, 130, and 131, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 132,133, and 134, respectively; or (x) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 135, 136, and 137, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 138,139, and 140, respectively.
 19. The method of claim 18, wherein theantibody or fragment thereof comprises a heavy chain CDR1, CDR2, andCDR3 comprising an amino acid sequence according to SEQ ID NOs: 93, 94,and 95, respectively; and a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence according to SEQ ID NOs: 96, 97, and 98,respectively.
 20. The method of claim 18, wherein the antibody orfragment thereof comprises a heavy chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence according to SEQ ID NOs: 81, 82, and 83,respectively; and a light chain CDR1, CDR2, and CDR3 comprising an aminoacid sequence according to SEQ ID NOs: 84, 85, and 86, respectively. 21.The method of claim 18, wherein the antibody or fragment thereof ishumanized.
 22. The method of claim 18, wherein the antibody or fragmentthereof comprises (i) a heavy chain variable region comprising SEQ IDNO: 2 and a light chain variable region comprising SEQ ID NO: 4; (ii) aheavy chain variable region comprising SEQ ID NO: 6 and a light chainvariable region comprising SEQ ID NO: 8; (iii) a heavy chain variableregion comprising SEQ ID NO: 10 and a light chain variable regioncomprising SEQ ID NO: 12; (iv) a heavy chain variable region comprisingSEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO:16; (v) a heavy chain variable region comprising SEQ ID NO: 18 and alight chain variable region comprising SEQ ID NO: 20; (vi) a heavy chainvariable region comprising SEQ ID NO: 22 and a light chain variableregion comprising SEQ ID NO: 24; (vii) a heavy chain variable regioncomprising SEQ ID NO: 26 and a light chain variable region comprisingSEQ ID NO: 28; (viii) a heavy chain variable region comprising SEQ IDNO: 30 and a light chain variable region comprising SEQ ID NO: 32; (ix)a heavy chain variable region comprising SEQ ID NO: 34 and a light chainvariable region comprising SEQ ID NO: 36; (x) a heavy chain variableregion comprising SEQ ID NO: 38 and a light chain variable regioncomprising SEQ ID NO: 40; (xi) a heavy chain variable region comprisingSEQ ID NO: 42 and a light chain variable region comprising SEQ ID NO:44; or (xii) a heavy chain variable region comprising SEQ ID NO: 46 anda light chain variable region comprising SEQ ID NO:
 48. 23. The methodof claim 18, wherein the antibody or fragment thereof comprises a heavychain variable region according to SEQ ID NO: 42 and a light chainvariable region according to SEQ ID NO:
 44. 24. The method of claim 18,wherein the antibody or fragment thereof comprises a heavy chainvariable region according to SEQ ID NO: 46 and a light chain variableregion according to SEQ ID NO:
 48. 25. The method claim 18, wherein theantibody or fragment thereof comprises a heavy chain according to SEQ IDNO: 70 and a light chain according to SEQ ID NO:
 74. 26. The method ofclaim 18, wherein the antibody or fragment thereof comprises a heavychain according to SEQ ID NO: 72 and a light chain according to SEQ IDNO:
 74. 27. The method of claim 18, wherein the antibody or fragmentthereof comprises a heavy chain according to SEQ ID NO: 76 and a lightchain according to SEQ ID NO:
 80. 28. The method of claim 1, wherein theantibody or fragment thereof comprises a heavy chain according to SEQ IDNO: 78 and a light chain according to SEQ ID NO:
 80. 29. The method ofclaim 18, wherein the antibody or fragment thereof is selected from thegroup consisting of a monoclonal antibody, an scFv, a Fab fragment, anFab′ fragment, and an F(ab)′ fragment.
 30. The method of claim 18,wherein the antibody or fragment thereof is linked or conjugated to atherapeutic agent.
 31. The method of claim 30, wherein the therapeuticagent is a cytotoxic drug, a radioactive isotope, an immunomodulator, oran antibody.
 32. The method of claim 18, wherein the method furthercomprises administering the antibody or fragment thereof to the subjectin combination with an additional therapeutic agent.
 33. A method fortreating an infectious disease in a subject in need thereof, the methodcomprising administering to the subject an antibody or fragment thereofthat binds to PD-L1, wherein the antibody or fragment thereof comprises:(i) a heavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:93, 94, and 95, respectively, and a light chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 96, 97, and 98, respectively; (ii) aheavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 81, 82,and 83, respectively, and a light chain CDR1, CDR2, and CDR3 sequencecomprising SEQ ID NO: 84, 85, and 86, respectively; (iii) a heavy chainCDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 87, 88, and 89,respectively, and a light chain CDR1, CDR2, and CDR3 sequence comprisingSEQ ID NO: 90, 91, and 92, respectively; (iv) a heavy chain CDR1, CDR2,and CDR3 sequence comprising SEQ ID NO: 99, 100, and 101, respectively,and a light chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:102, 103, and 104, respectively; (v) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 105, 106, and 107, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 108,109, and 110, respectively; (vi) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 111, 112, and 113, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 114,115, and 116, respectively; (vii) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 117, 118, and 119, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 120,121, and 122, respectively; (viii) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 123, 124, and 125, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 126,127, and 128, respectively; (ix) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 129, 130, and 131, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 132,133, and 134, respectively; or (x) a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 135, 136, and 137, respectively, and alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 138,139, and 140, respectively.
 34. The method of claim 33, wherein theinfectious disease is associated with PD-L1 expression on infectedcells.
 35. The method of claim 34, wherein the infectious disease isselected from the group consisting of candidiasis, candidemia,aspergillosis, streptococcal pneumonia, streptococcal skin andoropharyngeal conditions, gram negative sepsis, tuberculosis,mononucleosis, influenza, respiratory illness caused by RespiratorySyncytial Virus, malaria, schistosomiasis, and trypanosomiasis.